Synthesis of epothilones, intermediates thereto, analogues and uses thereof

ABSTRACT

The present invention provides convergent processes for preparing epothilone A and B, desoxyepothilones A and B, and analogues thereof. Also provided are analogues related to epothilone A and B and intermediates useful for preparing same. The present invention further provides novel compositions based on analogues of the epothilones and methods for the treatment of cancer and cancer which has developed a multidrug-resistant phenotype.

This application is a continuation of and claims priority under 35U.S.C. §120 to application Ser. No. 10/058,695, filed Jan. 28, 2002,which is a continuation of application Ser. No. 10/004,571, filed Dec.4, 2001, which is a continuation of application Ser. No. 09/874,514,filed Jun. 5, 2001, which is a continuation of application Ser. No.09/808,451, filed Mar. 14, 2001, now U.S. Pat. No. 6,656,961 which is acontinuation of application Ser. No. 08/986,025, filed Dec. 3, 1997, nowU.S. Pat. No. 6,242,469, issued Jun. 5, 2001, which claims priorityunder 35 U.S.C. §119(e) to U.S. Provisional Application Ser. Nos.60/032,282, 60/033,767, 60/047,566, 60/047,941, and 60/055,533, filedDec. 3, 1996, Jan. 14, 1997, May 22, 1997, May 29, 1997, and Aug. 13,1997, respectively, the contents of which are hereby incorporated byreference into this application.

This invention was made with government support under grants CA-28824,CA-39821, CA-GM 72231, CA-62948, and AIG9355 from the NationalInstitutes of Health, and grant CHE-9504805 from the National ScienceFoundation. Additionally, the present invention was supported in part bya fellowship from the United States Army to Dongfang Meng (DAMD17-97-1-7146), and thus the government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention is in the field of epothilone macrolides. Inparticular, the present invention relates to processes for thepreparation of epothilones A and B, desoxyepothilones A and B, andanalogues thereof which are useful as highly specific, non-toxicanticancer therapeutics. In addition, the invention provides methods ofinhibiting multidrug resistant cells. The present invention alsoprovides novel compositions of matter which serve as intermediates forpreparing the epothilones.

Throughout this application, various publications are referred to, eachof which is hereby incorporated by reference in its entirety into thisapplication to more fully describe the state of the art to which theinvention pertains.

BACKGROUND OF THE INVENTION

Epothilones A and B are highly active anticancer compounds isolated fromthe Myxobacteria of the genus Sorangium. The full structures of thesecompounds, arising from an x-ray crystallographic analysis weredetermined by Höfle. G. Höfle et al., Angew. Chem. Int. Ed. Engl., 1996,35, 1567. The total synthesis of the epothilones is an important goalfor several reasons. Taxol is already a useful resource in chemotherapyagainst ovarian and breast cancer and its range of clinicalapplicability is expanding. G. I. Georg et al., Taxane AnticancerAgents; American Cancer Society: San Diego, 1995. The mechanism of thecytotoxic action of taxol, at least at the in vitro level, involvesstabilization of microtubule assemblies. P. B. Schiff et al., Nature(London), 1979, 277, 665. A series of complementary in vitroinvestigations with the epothilones indicated that they share themechanistic theme of the taxoids, possibly down to the binding sites totheir protein target. D. M. Bollag et al., Cancer Res., 1995, 55, 2325.Moreover, the epothilones surpass taxol in terms of cytotoxicity and farsurpass it in terms of in vitro efficacy against drug resistant cells.Since multiple drug resistance (MDR) is one of the serious limitationsof taxol (L. M. Landino and T. L. MacDonald in The Chemistry andPharmacology of Taxol and its Derivatives, V. Farin, Ed., Elsevier: NewYork, 1995, ch. 7, p. 301), any agent which promises relief from thisproblem merits serious attention. Furthermore, formulating theepothilones for clinical use is more straightforward than taxol.

Accordingly, the present inventors undertook the total synthesis of theepothilones, and as a result, have developed efficient processes forsynthesizing epothilones A and B, the corresponding desoxyepothilones,as well as analogues thereof. The present invention also provides novelintermediates useful in the synthesis of epothilones A and B andanalogues thereof, compositions derived from such epothilones andanalogues, purified compounds of epothilones A and B, anddesoxyepothilones A and B, in addition to methods of use of theepothilone analogues in the treatment of cancer. Unexpectedly, certainepothilones have been found to be effective not only in reversingmulti-drug resistance in cancer cells, both in vitro and in vivo, buthave been determined to be active as collateral sensitive agents, whichare more cytotoxic towards MDR cells than normal cells, and assynergistic agents, which are more active in combination with othercytotoxic agents, such as vinblastin, than the individual drugs would bealone at the same concentrations. Remarkably, the desoxyepothilones ofthe invention have exceptionally high specificity as tumor cytotoxicagents in vivo, more effective and less toxic to normal cells than theprincipal chemotherapeutics currently in use, including taxol,vinblastin, adriamycin and camptothecin.

SUMMARY OF THE INVENTION

One object of the present invention is to provide processes for thepreparation of epothilones A and B, and desoxyepothilones A and B, andrelated compounds useful as anticancer therapeutics. Another object ofthe present invention is to provide various compounds useful asintermediates in the preparation of epothilones A and B as well asanalogues thereof.

A further object of the present invention is to provide syntheticmethods for preparing such intermediates. An additional object of theinvention is to provide compositions useful in the treatment of subjectssuffering from cancer comprising any of the analogues of the epothilonesavailable through the preparative methods of the invention optionally incombination with pharmaceutical carriers.

A further object of the invention is to provide methods of treatingsubjects suffering from cancer using any of the analogues of theepothilones available through the preparative methods of the inventionoptionally in combination with pharmaceutical carriers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1(A) shows a retrosynthetic analysis for epothilone A and B.

FIG. 1(B) provides synthesis of compound 11. (a) t-BuMe₂OTf, 2,6utidine,CH₂Cl₂, 98%; (b) (1) DDQ, CH₂Cl₂/H₂O, 89%; (2) (COCl)₂, DMSO, CH₂Cl₂,−78° C.; then Et₃N, −78° C.-rt, 90%; (c) MeOCH₂PPh₃Cl, t-BuOK, THF,0°-rt, 86%; (d) (1) p-TsOH, dioxane/H₂O, 50° C., 99%; (2) CH₃PPh₃Br,NaHMDS, PhCH₃, 0° C.-rt, 76%; (e) Phl(OCOCF₃)₂, MeOH/THF, rt, 0.25 h,92%.

FIG. 2 provides key intermediates in the preparation of 12,13-E- and-Z-deoxyepothilones.

FIGS. 3(A) and 3(B) provide syntheses of key iodinated intermediatesused to prepare hydroxymethylene- and hydroxypropylene-substitutedepothilone derivatives.

FIGS. 3(C) and 3(D) provide methods of preparing hydroxymethylene- andhydroxypropylene-substituted epothilone derivatives, said methods beinguseful generally to prepare 12,13-E epothilones wherein R is methyl,ethyl, n-propyl, and n-hexyl from the corresponding E-vinyl iodides.

FIGS. 3(E) and 3(F) show reactions leading to benzoylatedhydroxymethyl-substituted desoxyepothilone andhydroxymethylene-substituted epothilone (epoxide).

FIG. 4(A) provides synthesis of compound 19. (a) DHP, PPTS, CH₂Cl₂, rt:(b) (1) Me₃SiCCLi, BF₃.OEt₂, THF, −78° C.; (2) MOMCl, I-Pr₂NEt,Cl(CH₂)₂Cl, 55° C.; (3) PPTS, MeOH, rt; (c) (1) (COCl)₂, DMSO, CH₂Cl₂,−78° C.; then Et₃N, −78° C.-rt; (2) MeMgBr, Et₂O, 0° C.-rt, (3) TPAP,NMO, 4 Å mol. sieves, CH₂Cl₂, 0° C.-rt; (d) 16, n-BuLi, THF, −78° C.;then 15, THF, −78° C.-rt; (e) (1) N-iodosuccinimide, AgNO₃, (CH₃)₂CO;(2) Cy₂BH, Et₂O, AcOH; (f) (1) PhSH, BF₃.OEt₂, CH₂Cl₂, rt; (2) Ac₂O,pyridine, 4-DMAP, CH₂Cl₂, rt.

FIG. 4(B) presents synthesis of compound 1. (a) 11, 9-BBN, THF, rt; thenPdCl₂(dppf)₂, Cs₂CO₃, Ph₃As, H₂O, DMF, 19, rt, 71%; (b) p-TsOH,dioxane/H₂O, 50° C.; (c) KHMDS, THF, −78° C., 51%; (d) (1) HF-pyridine,pyridine, THF, rt, 97%; (2) t-BuMe₂ SiOTf, 2,6-lutidine, CH₂Cl₂, −25°C., 93%; (3) Dess-Martin periodinane, CH₂Cl₂, 87%; (4) HF-pyridine, THF,rt, 99%; (e) dimethyldioxirane, CH₂Cl₂, 0.5 h, −50° C., 45% (≧20:1).

FIG. 5 shows a scheme of the synthesis of the “left wing” of epothiloneA.

FIGS. 6(A) and 6(B) provide a scheme of an olefin metathesis route toepothilone A and other analogues.

FIG. 7 illustrates a convergent strategy for a total synthesis ofepothilone A (1) and the glycal cyclopropane solvolysis strategy for theintroduction of geminal methyl groups.

FIG. 8 provides an enantioselective synthesis of compound 15B.

FIG. 9 shows the construction of epothilone model systems 20B, 21B, and22B by ring-closing olefin metathesis.

FIG. 10 illustrates a sedimentation test for natural, synthetic anddesoxyepothilone A.

FIG. 11 illustrates a sedimentation test for natural, synthetic anddesoxyepothilone A after cold treatment at 4° C.

FIG. 12 illustrates (A) structures of epothilones A (1) and B (2) and(B) of Taxol™ (1A).

FIG. 13 shows a method of elaborating acyclic stereochemicalrelationships based on dihydropyrone matrices.

FIGS. 14(A) and 14(B) show the preparation of intermediate 4A.

FIG. 15 shows an alternative enantioselective synthesis of compound 17A.

FIG. 16 provides a synthetic pathway to intermediate 13C. (a) 1.tributyl allyltin, (S)-(−)-BINOL, Ti(Oi-Pr)₄, CH₂Cl₂, −20° C., 60%, >95%e.e.; 2. Ac₂O, Et₃N, DMAP, CH₂Cl₂, 95%; (b) 1. OsO₄, NMO, acetone/H₂O,0° C.; 2. NaIO₄, THF/H₂O; (c) 12, THF, −20° C., Z isomer only, 25% from10; (d) Pd(dppf)₂, Cs₂CO₃, Ph₃As H₂O, DMF, rt. 77%.

FIG. 17 provides a synthetic pathway to intermediate epothilone B (2).(a) p-TsOH, dioxane/H₂O, 55° C., 71%; (b) KHMDS, THF, −78° C., 67%,α/β1.5:1; (c) Dess-Martin periodinane, CH₂Cl₂; (d) NaBH₄, MeOH, 67% fortwo steps; (e) 1. HF.pyridine, pyridine, THF, rt, 93%; 2. TBSOTf,2,6-lutidine, CH₂Cl₂, −30° C., 89%; 3. Dess-Martin periodinane, CH₂Cl₂,67%; (f) HF.pyridine, THF, rt, 80%; (g) dimethyldioxirane, CH₂Cl₂, −50°C., 70%.

FIGS. 18(A) and 18(B) provide a synthetic pathway to a protectedintermediate for 8-desmethyl deoxyepothilone A.

FIGS. 19(A), 19(B) and 19(C) provide a synthetic pathway to 8-desmethyldeoxyepothilone A, and structures of trans-8-desmethyl-desoxyepothioloneA and a trans-iodoolefin intermediate thereto.

FIG. 20(A) shows structures of epothilones A and B and8-desmethylepothilone and FIG. 20(B) shows a synthetic pathway tointermediate TBS ester 10 used in the preparation of desmethylepothiloneA. (a) (Z)-Crotyl-B[(−)-Ipc]₂, −78° C., Et₂O, then 3N NaOH, 30% H₂O₂;(b) TBSOTf, 2,6-lutidine, CH₂Cl₂ (74% for two steps, 87% ee); (c) O₃,CH₂Cl₂/MeOH, −78° C., then DMS, (82%); (d) t-butyl isobutyrylacetate,NaH, BuLi, 0° C., then 6 (60%, 10:1); (e) Me₄NBH(OAc)₃, −10° C. (50%,10:1 α/β) or NaBH₄, MeOH, THF, 0° C., (88%, 1:1 α/β); (f) TBSOTf,2,6-lutidine, −40° C., (88%); (g) Dess-Martin periodinane, (90%); (h)Pd(OH)₂, H₂, EtOH (96%); (I) DMSO, oxalyl chloride, CH₂Cl₂, −78° C.(78%); (j) Methyl triphenylphosphonium bromide, NaHMDS, THF, 0° C.(85%); (k) TBSOTf, 2,6-lutidine, CH₂Cl₂, rt (87%).

FIG. 21 sews a synthetic pathway to 8-desmethylepothilone A. (a)Pd(dppf)₂Cl₂, Ph₃As, Cs₂CO, H₂O, DMF, rt (62%); (b) K₂CO₃, MeOH, H₂O(78%); (c) DCC, 4-DMAP, 4-DMAP.HCl, CHCl₃ (78%); (d) HF.pyr, THF, rt(82%), (e) 3,3-dimethyl dioxirane, CH₂Cl₂, −35° C. (72%, 1.5:1).

FIGS. 22(A), 22(B) and 22(C) show a synthetic pathway to prepareepothilone analogue 27D.

FIGS. 23(A), 23(B) and 23(C) show a synthetic pathway to prepareepothilone analogue 24D.

FIGS. 24(A) and 24(B) show a synthetic pathway to prepare epothiloneanalogue 19D.

FIGS. 25(A), 25(B), 25(C) and 25(D) show a synthetic pathway to prepareepothilone analogue 20D.

FIGS. 26(A), 26(B), 26(C) and 26(D) show a synthetic pathway to prepareepothilone analogue 22D.

FIGS. 27(A), 27(B) and 27(C) show a synthetic pathway to prepareepothilone analogue 12-hydroxy ethyl-epothilone.

FIGS. 28(A) and 28(B) show the activity of epothilone analogues in asedimentation test in comparison with DMSO, epothilone A and/or B.Structures 17-20, 22, and 24-27 are shown in FIGS. 29-37, respectively.Compounds were added to tubulin (1 mg/ml) to a concentration of 10 μM.The quantity of microtubules formed with epothilone A was defined as100%.

FIG. 29 shows a high resolution ¹H NMR spectrum of epothilone analogue#17.

FIG. 30 shows a high resolution ¹H NMR spectrum of epothilone analogue#18.

FIG. 31 shows a high resolution ¹H NMR spectrum of epothilone analogue#19.

FIG. 32 shows a high resolution ¹H NMR spectrum of epothilone analogue#20.

FIG. 33 shows a high resolution ¹H NMR spectrum of epothilone analogue#22.

FIG. 34 shows a high resolution ¹H NMR spectrum of epothilone analogue#24.

FIG. 35 shows a high resolution ¹H NMR spectrum of epothilone analogue#25.

FIG. 36 shows a high resolution ¹H NMR spectrum of epothilone analogue#26.

FIG. 37 shows a high resolution ¹H NMR spectrum of epothilone analogue#27.

FIG. 38 provides a graphical representation of the effect of fractionalcombinations of cytotoxic agents.

FIGS. 39(A) and 39(B) show epothilone A and epothilone analogues #1-7.Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBLMDR (resistant) sublines are shown in round and square brackets,respectively.

FIGS. 40(A) and 40(B) show epothilone B and epothilone analogues #8-16.Potencies against human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBLMDR (resistant) sublines are shown in round and square brackets,respectively.

FIGS. 41(A) and 41(B) show epothilone analogues #17-25. Potenciesagainst human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR(resistant) sublines are shown in round and square brackets,respectively.

FIGS. 42(A) and 42(B) show epothilone analogues #26-34. Potenciesagainst human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR(resistant) sublines are shown in round and square brackets,respectively.

FIGS. 42(C) and 42(D) show epothilone analogues #35-46. Potenciesagainst human leukemia CCRF-CEM (sensitive) and CCRF-CEM/VBL MDR(resistant) sublines are shown in round and square brackets,respectively.

FIG. 42(E) show epothilone analogues #47-49.

FIG. 43(A) shows antitumor activity of desoxyepothilone B against MDRMCF-7/Adr xenograft in comparison with taxol. Control (♦);desoxyepothilone B (▪; 35 mg/kg); taxol (▴; 6 mg/kg); adriamycin (×;1.8mg/kg); i.p. Q2Dx5; start on day 8.

FIG. 43(B) shows antitumor activity of epothilone B against MDRMCF-7/Adr xenograft in comparison with taxol. Control (♦); epothilone B(▪; 25 mg/kg; non-toxic dose); taxol (▴; 6 mg/kg; half LD₅₀); adriamycin(×;1.8 mg/kg); i.p. Q2Dx5; start on day 8.

FIG. 44(A) shows toxicity of desoxyepothilone B in B6D2F, mice bearingB16 melanoma. Body weight was determined at 0, 2, 4, 6, 8, 10 and 12days. Control (▴); desoxyepothilone B (∘; 10 mg/kg QDx8; 0 of 8 died);desoxyepothilone B (; 20 mg/kg QDx6; 0 of 8 died). Injections werestarted on day 1.

FIG. 44(B) shows toxicity of epothilone B in B6D2F, mice bearing B16melanoma. Body weight was determined at 0, 2, 4, 6, 8, 10 and 12 days.Control (▴); epothilone B (∘; 0.4 mg/kg QDx6; 1 of 8 died of toxicity);epothilone B (; 0.8 mg/kg QDx5; 5 of 8 died). Injections were startedon day 1.

FIG. 45w) shows comparative therapeutic effect of desoxyepothilone B andtaxol on nude mice bearing MX-1 xenoplant. Tumor, s.c.; drugadministered i.p., Q2Dx5, start on day 7. control (♦); Taxol (□; 5mg/kg, one half of LD₅₀); desoxyepothilone B (Δ; 25 mg/kg; nontoxicdose).

FIG. 45(B) shows comparative therapeutic effect of desoxyepothilone Band taxol on nude mice bearing MX-1 xenoplant. Tumor, s.c.; drugadministered i.p., Q2Dx5, start on day 7. control (♦); Taxol (□; 5mg/kg, one half of LD₅₀, given on days 7, 9, 11, 13, 15; then 6 mg/kg,given on days 17. 19, 23, 24, 25); desoxyepothilone B (n=3; Δ, ×, *; 25mg/kg, nontoxic dose, given to three mice on days 7, 9, 11, 13, 15; then35 mg/kg, given on days 17. 19, 23, 24, 25).

FIG. 46 shows the effect of treatment with desoxyepothilone B (35mg/kg), taxol (5 mg/kg) and adriamycin (2 mg/kg) of nude mice bearinghuman MX-1 xenograft on tumor size between 8 and 18 days afterimplantation. Desoxyepothilone B (□), taxol (Δ), adriamycin (×), control(♦); i.p. treatments were given on day 8, 10, 12, 14 and 16.

FIG. 47 shows the relative toxicity of epothilone B (□; 0.6 mg/kg QDx4;i.p.) and desoxyepothilone B (Δ; 25 mg/kg QDx4; i.p.) versus control (♦)in normal nude mice. Body weight of mice was determined daily afterinjection. For epothilone B, 8 of 8 mice died of toxicity on days 5, 6,6, 7, 7, 7, 7, and 7; for desoxyepothilone B, all six mice survived.

FIG. 48 shows a high resolution ¹H NMR spectrum of epothilone analogue#43.

FIG. 49 shows a high resolution ¹H NMR spectrum of epothilone analogue#45.

FIG. 50 shows a high resolution ¹H NMR spectrum of epothilone analogue#46.

FIG. 51 shows a high resolution ¹H NMR spectrum of epothilone analogue#47.

FIG. 52 shows a high resolution ¹H NMR spectrum of epothilone analogue#48.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “linear or branched chain alkyl” encompasses,but is not limited to, methyl, ethyl, propyl, isopropyl, t-butyl,sec-butyl, cyclopentyl or cyclohexyl. The alkyl group may contain onecarbon atom or as many as fourteen carbon atoms, but preferably containsone carbon atom or as many as nine carbon atoms, and may be substitutedby various groups, which include, but are not limited to, acyl, aryl,alkoxy, aryloxy, carboxy, hydroxy, carboxamido and/or N-acylaminomoieties.

As used herein, the terms “alkoxycarbonyl”, “acyl” and “alkoxy”encompass, but are not limited to, methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, n-butoxycarbonyl, benzyloxycarbonyl,hydroxypropylcarbonyl, aminoethoxycarbonyl, sec-butoxycarbonyl andcyclopentyloxycarbonyl. Examples of acyl groups include, but are notlimited to, formyl, acetyl, propionyl, butyryl and penanoyl. Examples ofalkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy,n-butoxy, sec-butoxy and cyclopentyloxy.

As used herein, an “aryl” encompasses, but is not limited to, a phenyl,pyridyl, pyrryl, indolyl, naphthyl, thiophenyl or furyl group, each ofwhich may be substituted by various groups, which include, but are notlimited, acyl, aryl alkoxy, aryloxy, carboxy, hydroxy, carboxamido orN-acylamino moieties. Examples of aryloxy groups include, but are notlimited to, a phenoxy, 2-methylphenoxy, 3-methylphenoxy and 2-naphthoxy.Examples of acyloxy groups include, but are not limited to, acetoxy,propanoyloxy, butyryloxy, pentanoyloxy and hexanoyloxy.

The subject invention provides chemotherapeutic analogues of epothiloneA and B, including a compound having the structure:

wherein R, R₀ and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched alkyl; and wherein n is 0, 1, 2, or 3. In oneembodiment, the invention provides the compound having the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH₂OH, or(CH₂)₃OH.

The invention also provides a compound having the structure:

wherein R, R₀, and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or 3. In acertain embodiment, the invention provides a compound having thestructure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl or CH₂OH.

In addition, the invention provides a compound having the structure.

wherein R, R₀, and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino, or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2, or 3. Inparticular, the invention provides a compound having the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, CH₂OH or (CH₂)₃OH.

The invention further provides a compound having the structure:

wherein R, R₀ and R′ are independently H, linear or branched chainalkyl, optionally substituted by hydroxy, alkoxy, fluorine, NR₁R₂,N-hydroximino or N-alkoxyimino, wherein R₁ and R₂ are independently H,phenyl, benzyl, linear or branched chain alkyl; wherein R″ is —CHY═CHX,or H, linear or branched chain alkyl, phenyl, 2-methyl-1,3-thiazolinyl,2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or 6-indolyl; and whereinX is H, linear or branched chain alkyl, phenyl,2-methyl-1,3-thiazolinyl, 2-furanyl, 3-furanyl, 4-furanyl, 2-pyridyl,3-pyridyl, 4-pyridyl, imidazolyl, 2-methyl-1,3-oxazolinyl, 3-indolyl or6-indolyl; wherein Y is H or linear or branched chain alkyl; wherein Zis O, N(OR₃) or N—NR₄R₅, wherein R₃, R₄ and R₅ are independently H or alinear or branched chain alkyl; and wherein n is 0, 1, 2 or 3.

The invention also provides a compound having the structure:

The subject invention also provides various inter mediates useful forthe preparation of the chemotherapeutic compounds epithilone A and B, aswell as analogues thereof. Accordingly, the invention provides a keyintermediate to epothilone A and its analogues having the structure:

wherein R is hydrogen, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; wherein R′ is hydrogen, methyl, ethyl,n-propyl, n-hexyl,

CH₂OTBS or (CH₂)₃-OTBDPS; and X is a halide. In one embodiment, thesubject invention provides a compound of the above structure wherein Ris acetyl and X is iodo.

The subject invention also provides an intermediate having thestructure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR)₂, (SR)₂, —(O—(CH₂)_(n)—O)—, —(O(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S)—;and wherein n is 2, 3 or 4.

wherein R is H or methyl.

Another analogue provided by the invention has the structure:

wherein R is H, methyl, ethyl, n-propyl, n-butyl, n-hexyl, CH₂OH, or(CH₂)₃OH.

Additionally, the subject invention provides in analogue having thestructure:

wherein R is H or methyl. The scope of the present invention includescompounds wherein the C₃ carbon therein possesses either an R or Sabsolute configuration, as well as mixtures thereof.

The subject invention further provides an analogue of epothilone Ahaving the structure:

The subject invention also provides synthetic routes to prepare theintermediates for preparing epothilones. Accordingly, the inventionprovides a method of preparing a Z-iodoalkene ester having thestructure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalky,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises (a)coupling a compound having the structure:

with a methyl ketone having the structure:

wherein R′ and R″ are independently a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryl or benzyl, under suitableconditions to form a compound having the structure:

(b) treating the compound formed in step (a) under suitable conditionsto form a Z-iodoalkene having the structure:

and (c) deprotecting and acylating the Z-iodoalkene formed in step (b)under suitable conditions to form the Z-iodoalkene ester. The couplingin stem) (a) may be effected using a strong base such as n-BuLi in aninert polar solvent such as tetrahydrofuran (THF) at low temperatures,typically below −50° C., and preferably at −78° C. The treatment in step(b) may comprise sequential reaction with N-iodosuccinimide in thepresence of Ag(I), such as silver nitrate, in a polar organic solventsuch as acetone, followed by reduction conditions, typically using ahydroborating reagent, preferably using Cy₂BH. Deprotecting step (c)involves contact with a thiol such as thiophenol in the presence of aLevis acid catalyst, such as boron trifluoride-etherate in an inertorganic solvent such as dichloromethane, followed by acylation with anacyl halide, such as acetyl chloride, or an acyl anhydride, such asacetic anhydride in the presence of a mild base such as pyridine and/or4-dimethylaminopyridine (DMAP) in an inert organic solvent such asdichloromethane.

The subject invention also provides a method of preparing a Z-haloalkeneester having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; and wherein X is ahalogen, which comprises (a) oxidatively cleaving a compound having thestructure:

under suitable conditions to form an aldehyde intermediate; and (b)condensing the aldehyde intermediate with a halomethylene transfer agentunder suitable conditions to form the Z-haloalkene ester. In oneembodiment of the method, X is iodine. In another embodiment, the methodis practiced wherein the halomethylene transfer agent is Ph₃P═CHl or(Ph₃P⁺CH₂I)I⁻. Disubstituted olefins may be prepared using thehaloalkylidene transfer agent Ph₃P═CR′I, wherein R′ is hydrogen, methyl,ethyl, n-propyl, n-hexyl,

CO₂Et or (CH₂)₃OTBDPS. The oxidative step (a) can be performed using amild oxidant such as osmium tetraoxide at temperatures of about 0° C.,followed by treatment with sodium periodate, or with leadtetraacetatelsodium carbonate, to complete the cleavage of the terminalolefin, and provide a terminal aldehyde. Condensing step (b) occurseffectively with a variety of halomethylenating reagents, such as Wittigreagents.

The subject invention further provides a method of preparing anoptically pure compound having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)condensing an allylic organometallic reagent with an unsaturatedaldehyde having the structure:

under suitable conditions to form an alcohol, and, optionallyconcurrently therewith, optically resolving the alcohol to form anoptically pure alcohol having the structure:

(b) alkylating or acylating the optically pure alcohol formed in step(a) under suitable conditions to form the optically pure compound. Inone embodiment of the method, the allylic organometallic reagent is anallyl(trialkyl)stannane. In another embodiment, the condensing step iseffected using a reagent comprising a titanium tetraalkoxide and anoptically active catalyst. In step (a) the 1,2-addition to theunsaturated aldehyde may be performed using a variety of allylicorganometallic reagents, typically with an allyltrialkylstannane, andpreferably with allyltri-n-butylstannane, in the presence of chiralcatalyst and molecular sieves in an inert organic solvent such asdichloromethane. Preferably, the method may be practiced using titaniumtetraalkoxides, such as titanium tetra-n-propoxide, and S(−)BINOL as theoptically active catalyst. Alkylating or acylating step (b) is effectedusing any typical alkylating agent, such as alkylhalide or alkyltosylate, alkyl triflate or alkyl mesylate, any typical acylating agent,such as acetyl chloride, acetic anhydride, benzoyl chloride or benzoylanhydride, in the presence of a mild base catalyst in an inert organicsolvent, such as dichloromethane.

The subject invention also provides a method of preparing an open-chainaldehyde having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises: (a)cross-coupling a haloolefin having the structure:

wherein R is a linear or branched alkyl, alkoxyalkyl, substituted orunsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, and X is a halogen, with a terminalolefin having the structure:

wherein (OR′″)₂, is (OR₀)₂, (SR₀)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)—or —(S—(CH₂)_(n)—S)— where R₀ is a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl; and wherein n is 2, 3 or 4, undersuitable conditions to form a cross-coupled compound having thestructure:

wherein Y is CH(OR*)₂ where R* is a linear or branched alkyl,alkoxyalkyl, substituted or unsubstituted aryloxyalkyl; and (b)deprotecting the cross-coupled compound formed in step (a) undersuitable conditions to form the open-chain compound. Cross-coupling step(a) is effected using reagents known in the art which are suited to thepurpose. For example, the process may be carried out by hydroboratingthe pre-acyl component with 9-BBN. The resulting mixed borane may thenbe cross-coupled with an organometallic catalyst such as PdCl₂(dppf)₂,or any known equivalent thereof, in the presence of such ancillaryreagents as cesium carbonate and triphenylarsine. Deprotecting step (b)can be carried out with a mild acid catalyst such as p-tosic acid, andtypically in a mixed aqueous organic solvent system, such asdioxane-water. The open-chain compound can be cyclized using any of avariety of non-nucleophilic bases, such as potassiumhexamethyldisilazide or lithium diethyamide.

The subject invention also provides a method of preparing an epothilonehaving the structure:

which comprises: (a) deprotecting a cyclized compound having thestructure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, under suitable conditionsto form a deprotected cyclized compound and oxidizing the deprotectedcyclized compound under suitable conditions to form a desoxyepothilonehaving the structure.

and (b) epoxidizing the desoxyepothilone formed in step (a) undersuitable conditions to form the epothilone. Deprotecting step (a) iseffected using a sequence of treatments comprising a catalyst such asHF-pyridine, followed by t-butyldimethylsilyl triflate in the presenceof a base such as lutidine. Dess-Martin oxidation and furtherdeprotection with a catalyst such as HF-pyridine provides thedesoxyepothilone. The latter compound can then be epoxidized in step (b)using any of a variety of epoxidizing agents, such acetic peracid,hydrogen peroxide, perbenzoic acid, m-chloroperbenzoic acid, butpreferably with dimethyldioxirane, in an inert organic solvent such asdichloromethane.

The subject invention further provides a method of preparing anepothilone precursor having the structure:

wherein R₁ is hydrogen or methyl; wherein X is O, or a hydrogen and OR″,each singly bonded to carbon; and wherein R₀, R′ and R″ areindependently hydrogen, a linear or branched alkyl, substituted orunsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises (a) coupling a compoundhaving the structure:

wherein R is an acetyl, with an aldehyde having the structure:

wherein Y is oxygen, under suitable conditions to form an aldolintermediate and optionally protecting the aldol intermediate undersuitable conditions to form an acyclic epthilone precursor having thestructure:

(b) subjecting the acylic epothilone precursor to conditions leading tointramolecular olefin metathesis to form the epothilone precursor. Inone embodiment of the method, the conditions leading to intramolecularolefin metathesis require the presence of an organometallic catalyst. Ina certain specific embodiment of the method, the catalyst contains Ru orMo. The coupling step (a) may be effected using a nonnucleophilic basesuch as lithium diethylamide or lithium diisopropylamide at subambienttemperatures, but preferably at about −78° C. The olefin metathesis instep (b) may be carried out using any catalyst known in the art suitedfor the purpose, though preferably using one of Grubbs's catalysts.

In addition, the present invention provides a compound useful as anintermediate for preparing epothilones having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR*)₂, (SR*)₂, —(O—(CH₂)_(n)—O—, —(O—(CH₂)_(n)—S)— or—(S—(CH₂)_(n)—S)—; wherein R* is a linear or branched alkyl, substitutedor unsubstituted aryl or benzyl; wherein R₂B is a linear, branched orcyclic boranyl moiety-, and wherein n is 2, 3 or 4. In certainembodiments, the invention provides the compound wherein R′ is TBS, R″is TPS and X is (OMe)₂. A preferred example of R₂B is derived from9-BBN.

The invention also provides the compound having the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein X is oxygen,(OR)₂, (SR)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or —(S—(CH₂)_(n)—S—;and wherein n is 2, 3 or 4. In certain embodiments, the inventionprovides the compound wherein R′ is TBS, R″ is TPS and X is (OMe)₂.

The invention further provides a desmethylepothilone analogoue havingthe structure:

wherein R is H or methyl.

The invention provides a compound having the structure:

wherein R is H or methyl.

The invention also provides a trans-desmethyldeoxyepothilone analoguehaving the structure:

wherein R is H or methyl.

The invention also provides a trans-epothilone having the structure:

wherein R is H or methyl.

The invention also provides a compound having the structure:

wherein R is hydrogen, a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryloxyalkyl, linear or branched acyl,substituted or unsubstituted aroyl or benzoyl; wherein R′ is hydrogen,methyl, ethyl, n-propyl, n-hexyl,

CO₂Et or (CH₂)₃OTBDPS. and X is a halogen. In certain embodiments, theinvention provides the compound wherein R is acetyl and X is iodine.

The invention additionally provides a method of preparing an open-chainaldehyde having the structure:

wherein R is a linear or branched alkyl, alkoxyalkyl, substituted orunsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R′ and R″ are independentlyhydrogen, a linear or branched alkyl, substituted or unsubstituted arylor benzyl, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linearor branched acyl, substituted or unsubstituted aroyl or benzoyl, whichcomprises:

(a) cross-coupling a haloolefin having the structure:

 wherein X is a halogen, with a terminal borane having the structure:

 wherein R*₂B is a linear, branched or cyclic alkyl or substituted orunsubstituted aryl or benzyl boranyl moiety; and wherein Y is (OR₀)₂,(SR)₂, —(O—(CH₂)_(n)—O)—, —(O—(CH₂)_(n)—S)— or S—(CH₂)_(n)—S)— where R₀is a linear or branched alkyl, substituted or unsubstituted aryl orbenzyl; and wherein n is 2, 3 or 4, under suitable conditions to form across-coupled compound having the structure:

 and

(b) deprotecting the cross-coupled compound formed in step (a) undersuitable conditions to form the open-chain aldehyde. In certainembodiments, the invention provides the method wherein R is acetyl; R′is TBS; R″ is TPS; R*₂B is derived from 9-BBN; and Y is (OMe)₂.Cross-coupling step (a) is effected using reagents known in the artwhich are suited to the purpose. For example, the mixed borane may becross-coupled with an organometallic catalyst such as PdCl₂(dppf)₂, orany known equivalent thereof, in the presence of such reagents as cesiumcarbonate and triphenylarsine. Deprotecting step (b) can be carried outusing a mild acid catalyst such as p-tosic acid, typically in a mixedaqueous organic solvent system, such as dioxane-water.

The invention also provides a method of preparing a protected epothilonehaving the structure:

wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkyl-arylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, which comprises:

(a) monoprotecting a cyclic diol having the structure:

 under suitable conditions to form a cyclic alcohol having thestructure:

 and

(b) oxidizing the cyclic alcohol formed in step (a) under suitableconditions to form the protected epothilone. In certain embodiments, theinvention provides the method wherein R′ and R″ are TBS. Themonoprotecting step (a) may be effected using any of a variety ofsuitable reagents, including TBSOTf in the presence of a base in aninert organic solvent. The base may be a non-nucleophilic base such as2,6-lutidine, and the solvent may be dichloromethane. The reaction isconducted at subambient temperatures, preferably in the range of −30° C.The oxidizing step (b) utilizes a selective oxidant such as Dess-Martinperiodinane in an inert organic solvent such as dichloromethane. Theoxidation is carried out at ambient temperatures, preferably at 20-25°C.

The invention further provides a method of preparing an epothilonehaving the structure:

which comprises:

(a) deprotecting a protected cyclic ketone having the structure:

 wherein R′ and R″ are independently hydrogen, a linear or branchedalkyl, substituted or unsubstituted aryl or benzyl, trialkylsilyl,dialkylarylsilyl, alkyldiarylsilyl, a linear or branched acyl,substituted or unsubstituted aroyl or benzoyl, under suitable conditionsto form a desoxyepothilone having the structure:

 and

(b) epoxidizing the desoxyepothilone formed in step (a) under suitableconditions to form the epothilone. In certain embodiments, the inventionprovides the method wherein R′ and R″ are TBS. Deprotecting step (a) iscarried out by means of a treatment comprising a reagent such asHF-pyridine. The deprotected compound can be epoxidized in step (b)using an epoxidizing agent such acetic peracid, hydrogen peroxide,perbenzoic acid, m-chloroperbenzoic acid, but preferably withdimethyldioxirane, in an inert organic solvent such as dichloromethane.

The invention also provides a method of preparing a cyclic diol havingthe structure:

wherein R′ is a hydrogen, a linear or branched alkyl, substituted orunsubstituted aryl or benzyl, trialkylsilyl, dialkylarylsilyl,alkyldiarylsilyl, a linear or branched acyl, substituted orunsubstituted aroyl or benzoyl, which comprises:

(a) cyclizing an open-chain aldehyde having the structure:

 wherein R is a linear or branched alkyl, alkoxyalkyl, substituted orunsubstituted aryloxyalkyl, trialkylsilyl, aryldialkylsilyl,diarylalkylsilyl, triarylsilyl, linear or branched acyl, substituted orunsubstituted aroyl or benzoyl; and wherein R″ is a hydrogen, a linearor branched alkyl, substituted or unsubstituted aryl or benzyl,trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, a linear or branchedacyl, substituted or unsubstituted aroyl or benzoyl under suitableconditions to form an enantiomeric mixture of a protected cyclic alcoholhaving the structure:

 said mixture comprising an α- and a β-alcohol component;

(b) optionally isolating and oxidizing the a-alcohol formed in step (a)under suitable conditions to form a ketone and thereafter reducing theketone under suitable conditions to form an enantiomeric mixture of theprotected cyclic alcohol comprising substantially the β-alcohol; and

(c) treating the protected cyclic alcohol formed in step (a) or (b) witha deprotecting agent under suitable conditions to form the cyclic diol.In certain embodiments, the invention provides the method wherein R′ isTBS and R″ is TPS. Cyclizing step (a) is performed using any of avariety of mild nonnucleophilic bases such as KHMDS in an inert solventsuch as THF. The reaction is carried out at subambient temperatures,preferably between −90° C. and −50° C., more preferably at −78° C.Isolation of the unnatural alpha-OH diastereomer is effected by anypurification method, including any suitable type of chromatography or bycrystallization. Chromatographic techniques useful for the purposeinclude high pressure liquid chromatography, countercurrentchromatography or flash chromatography. Various column media are suited,including, inter alia, silica or reverse phase support. The beta-OHderivative is then oxidized using a selective oxidant, such asDess-Martin periodinane. The resulting ketone is the reduced using aselective reductant. Various hydridoborane and aluminum hydride reagentsare effective. A preferred reducing agent is sodium borohydride.Treating step (c) may be effected using a variety of deprotectingagents, including HF-pyridine.

In addition, the invention provides a method of treating cancer in asubject suffering therefrom comprising administering to the subject atherapeutically effective amount of any of the analogues related toepothilone B disclosed herein optionally in combination with apharmaceutically suitable carrier. The method may be applied where thecancer is a solid tumor or leukemia. In particular, the method isapplicable where the cancer is breast cancer or melanoma.

The subject invention also provides a pharmaceutical composition fortreating cancer comprising any of the analogues of epothilone disclosedhereinabove, as an active ingredient, optionally though typically incombination with a pharmaceutically suitable carrier. The pharmaceuticalcompositions of the present invention may further comprise othertherapeutically active ingredients.

The subject invention further provides a method of treating cancer in asubject suffering therefrom comprising administering to the subject atherapeutically effective amount of any of the analogues of epothilonedisclosed hereinabove and a pharmaceutically suitable carrier. Themethod is especially useful where the cancer is a solid tumor orleukemia.

The compounds taught above which are related to epothilones A and B areuseful in the treatment of cancer, and particularly, in cases wheremultidrug resistance is present, both in vivo and in vitro. The abilityof these compounds as non-substrates of MDR in cells, as demonstrated inthe Tables below, shows that the compounds are useful to treat, preventor ameliorate cancer in subjects suffering therefrom.

The magnitude of the therapeutic dose of the compounds of the inventionwill vary with the nature and severity of the condition to be treatedand with the particular compound and its route of administration. Ingeneral, the daily dose range for anticancer activity lies in the rangeof 0.001 to 25 mg/kg of body weight in a mammal, preferably 0.001 to 10mg/kg, and most preferably 0.001 to 1.0 mg/kg, in single or multipledoses. In unusual cases, it may be necessary to administer doses above25 mg/kg.

Any suitable route of administration may be employed for providing amammal, especially a human, with an effective dosage of a compounddisclosed herein. For example, oral, rectal, topical, parenteral,ocular, pulmonary, nasal, etc., routes may be employed. Dosage formsinclude tablets, troches, dispersions, suspensions, solutions, capsules,creams, ointments, aerosols, etc.

The compositions include compositions suitable for oral, rectal, topical(including transdermal devices, aerosols, creams, ointments, lotions anddusting powders), parenteral (including subcutaneous, intramuscular andintravenous), ocular (ophthalmic), pulmonary (nasal or buccalinhalation) or nasal administration. Although the most suitable route inany given case will depend largely on the nature and severity of thecondition being treated and on the nature of the active ingredient. Theymay be conveniently presented in unit dosage form and prepared by any ofthe methods well known in the art of pharmacy.

In preparing oral dosage forms, any of the unusual pharmaceutical mediamay be used, such as water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, and the like in the case of oral liquidpreparations (e g., suspensions, elixers and solutions); or carrierssuch as starches, sugars, microcrystalline cellulose, diluents,granulating agents, lubricants, binders, disintegrating agents, etc., inthe case of oral solid preparations are preferred over liquid oralpreparations such as powders, capsules and tablets. If desired, capsulesmay be coated by standard aqueous or non-aqueous techniques. In additionto the dosage forms described above, the compounds of the invention maybe administered by controlled release means and devices.

Pharmaceutical compositions of the present invention suitable for oraladministration may be prepared as discrete units such as capsules,cachets or tablets each containing a predetermined amount of the activeingredient in powder or granular form or as a solution or suspension inan aqueous or nonaqueous liquid or in an oil-in-water or water-in-oilemulsion. Such compositions may be prepared by any of the methods knownin the art of pharmacy. In general compositions are prepared byuniformly and intimately admixing the active ingredient with liquidcarriers, finely divided solid carriers, or both and then, if necessary,shaping the product into the desired form. For example, a tablet may beprepared by compression or molding, optionally with one or moreaccessory ingredients. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas powder or granule optionally mixed with a binder, lubricant, inertdiluent or surface active or dispersing agent. Molded tablets may bemade by molding in a suitable machine, a mixture of the powderedcompound moistened with an inert liquid diluent.

The present invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described in the claims which followthereafter. It will be understood that the processes of the presentinvention for preparing epothilones A and B, analogues thereof andintermediates thereto encompass the use of various alternate protectinggroups known in the art. Those protecting groups used in the disclosureincluding the Examples below are merely illustrative.

EXAMPLE 1

THP glycidol 13:

A solution of (R)-(+)-glycidol 12 (20 g; 270 mmol) and freshly distilled3,4-dihydro-2H-pyran (68.1 g; 810 mmol) in CH₂Cl₂ (900 ml) was treatedwith pyridinium p-toluenesulfonate (2.1 g; 8.36 mmol) at rt and theresulting solution was stirred for 16 h. Approximately 50% of thesolvent was then removed in vacuo and the remaining solution was dilutedwith ether (1 L). The organic layer was then washed with two portions ofsaturated aqueous sodium bicarbonate (500 ml), dried (Na₂SO₄), filtered,and concentrated. Purification of the residue by flash chromatography(silica, 25→50% ether hexanes) afforded THP glycidol 13 (31.2 g; 73%) asa colorless liquid: IR(film): 2941, 1122, 1034 cm⁻¹; ¹H NMR(CDCl₃, 500MHz) d 4.66(t, J=3.5 Hz, 1 H), 4.64 (t, J=3.5 Hz, 1H), 3.93 (dd, J=11.7,3.1 Hz, 1H), 3.86 (m, 2H), 3.73 (dd, J=11.8, 5.03 Hz, 1H), 3.67 (dd,J=11.8, 3.4 Hz, 1H), 3.51 (m, 2H), 3.40 (dd, J=11.7, 6.4, 1H), 3.18 (m,2H), 2.80 (m, 2H), 2.67 (dd, J=5.2, 2.7 Hz, 1H), 2.58 (dd, J=5.0, 2.7Hz, 1H), 1.82 (m, 2H), 1.73 (m, 2H), 1.52 (m, 4H); ¹³C NMR (CDCl₃, 125MHz) d 98.9, 98.8, 68.5, 67.3, 62.4, 62.2, 50.9, 50.6, 44.6, 44.5, 30.5,30.4, 25.4, 19.3, 19.2; [α]_(D)=+4.98 (c=2.15, CHCl₃).

EXAMPLE 2

Alcohol 13a:

Trimethylsilylacetylene (32.3 g; 329 mmol) was added via syringe to THF(290 ml), and the resulting solution was cooled to −78° C. and treatedwith n butyllithium (154 ml of a 1.6 M solution in hexanes; 246.4 mmol).After 15 min, boron trifluoride diethyl etherate (34.9 g; 246 mmol) wasadded, and the resulting mixture was stirred for 10 min. A solution ofepoxide 13 (26 g; 164.3 mmol) in THF (130 ml) was then added via acannula and the resulting solution was stirred for 5.5 h at −78° C. Thereaction was quenched by the addition of saturated aqueous sodiumbicarbonate solution (250 ml) and the solution was allowed to warm tort. The mixture was then diluted with ether (600 ml) and washedsuccessively with saturated aqueous sodium bicarbonate solution (250ml), water (250 ml), and brine (250 ml). The organic layer was thendried (Na₂SO₄), filtered, and concentrated in vacuo. Purification of theresidue by flash chromatography (silica, 20% ether:hexanes) providedalcohol 13a (34 g; 76%).

EXAMPLE 3

MOM ether 13b:

A solution of alcohol 13a (24 g; 88.9 mmol) andN,N-diisopropylethylamine (108 ml; 622 mmol) in anhydrous1,2-dichloroethane (600 ml) was treated with chloromethyl methyl ether(17 ml, 196 mmol), and the resulting mixture was heated to 55° C. for 28h. The dark mixture was then cooled to rt and treated with saturatedaqueous sodium bicarbonate solution (300 ml). The layers were separated,and the organic layer was washed successively with saturated aqueoussodium bicarbonate solution (200 ml) and brine (200 ml). The organiclayer was then dried (MgSO₄) and filtered through a pad of silica gel(ether rinse). Purification of the residue by flash chromatography(silica, 20-30% ether:hexanes) afforded MOM ether 13b (23.7 g; 85%) as apale yellow oil.

EXAMPLE 4

Alcohol 14:

A solution of THP ether 13b (20 g; 63.7 mmol) in methanol (90 ml) wastreated with pyridinium p-toluenesulfonate (4.0 g; 15.9 mmol) and theresulting mixture was stirred at rt for 16 h. The reaction was thenquenched by the addition of saturated aqueous sodium bicarbonatesolution (100 ml), and the excess methanol was removed in vacuo. Theresidue was diluted with ether (300 ml), and the organic layer waswashed successively with saturated aqueous sodium bicarbonate solution(200 ml) and brine (200 ml). The organic layer was dried (MgSO₄),filtered, and concentrated. Purification of the residue by flashchromatography (silica, 40-50% ether:hexanes) provided alcohol 14 (13.1g; 95%) as a colorless oil.

EXAMPLE 5

Alcohol 14a:

To a cooled (−78° C.) solution of oxalyl chloride (24.04 ml of a 2.0 Msolution in CH₂Cl₂; 48.08 mmol) in CH₂Cl₂ (165 ml) was added anhydrousDMSO (4.6 ml; 64.1 mmol in dropwise fashion. After 30 min, a solution ofalcohol 14 (6.93 g; 32.05 mmol) in CH₂Cl₂ (65 ml+10 ml rinse) was addedand the resulting solution was stirred at −78° C. for 40 min. Freshlydistilled triethylamine (13.4 ml; 96.15 mmol) was then added, thecooling bath was removed, and the mixture was allowed to warm to 0° C.The reaction mixture was then diluted with ether (500 ml), and washedsuccessively with two portions of water (250 ml) and one portion ofbrine (250 ml). The organic layer was then dried (MgSO₄), filtered, andconcentrated.

The crude aldehyde (6.9 g) prepared in the above reaction was dissolvedin ether (160 ml) and cooled to 0° C. Methylmagnesium bromide (32.1 mlof a 3.0 M solution in butyl ether; 96.15 mmol) was then added, and thesolution was allowed to warm slowly to rt. After 10 h, the reactionmixture was cooled to 0° C. and the reaction was quenched by theaddition of saturated aqueous ammonium chloride solution. The mixturewas diluted with ether (200 ml) and washed successively with water (150ml) and brine (150 ml). The organic layer was dried (MgSO₄), filtered,and concentrated. Purification of the residue by flash chromatography(silica, 40-50% ether:hexanes) provided alcohol 14a (6.3 g; 85% from14).

EXAMPLE 6

Ketone 15:

A solution of alcohol 14 (1.0 g; 4.35 mmol), 4 Å mol. sieves, andN-methylmorpholine-N-oxide (1.0 g; 8.7 mmol) in CH₂Cl₂ (20 ml) at rt wastreated with a catalytic amount of tetra-n-propylammonium perruthenate,and the resulting black suspension was stirred for 3 h. The reactionmixture was then filtered through a pad of silica gel (ether rinse), andthe filtrate was concentrated in vacuo. Purification of the residue byflash chromatography (silica, 10% ether:hexanes) afforded ketone 15 (924mg; 93%) as a light yellow oil.

EXAMPLE 7

Alkene 17:

A cooled (−78° C.) solution of phosphine oxide 16 (1.53 g; 4.88 mmol) inTHF (15.2 ml) was treated with n-butyllithium (1.79 ml of a 2.45 Msolution in hexanes). After 15 min, the orange solution was treated witha solution of ketone 15 (557 mg; 2.44 mmol) in THF (4.6 ml). After 10min, the cooling bath was removed, and the solution was allowed to warmto rt. The formation of a precipitate was observed as the solutionwarmed. The reaction was quenched by the addition of saturated aqueousammonium chloride solution (20 ml). The mixture was then poured intoether (150 ml) and washed successively with water (50 ml) and brine (50ml). The organic layer was dried (Na₂SO₄), filtered, and concentrated.Purification of the residue by flash chromatography (silica, 10%ether:hexanes) afforded alkene 17 (767 mg; 97%) as a colorless oil:IR(film): 2956, 2177, 1506, 1249, 1149, 1032, 842, cm⁻¹; ¹H NMR(CDCl₃,500 MHz) d 6.95(s, 1H), 6.53(s, 1H), 4.67(d, J=6.7 Hz, 1H), 4.57 (d,J=6.8 Hz, 1H), 4.29 (dd, J=8.1, 5.4 Hz, 1H), 3.43 (s, 3H), 2.70 (s, 3H),2.62 (dd, J=16.9, 8.2 Hz, 1H), 2.51 (dd, J=17.0, 5.4 Hz, 1H), 2.02(s,3H); ¹³C NMR (CDCl₃, 125 MHz) d 164.4, 152.5, 137.1, 121.8, 116.2,103.7, 93.6, 86.1, 79.6, 55.4, 25.9, 19.1, 13.5; [α]_(D)=−27.3 (c=2.2,CHCl₃).

EXAMPLE 8

Alkenyl iodide formation:

To a solution of the alkyne 17 (3.00 g, 9.29 mmol) in acetone (100 mL)at 0° C. was added NIS (2.51 g; 11.2 mmol) and AgNO₃ (0.160 g; 0.929mmol). The mixture was then slowly warmed to rt. After 1.5 h, thereaction was poured into Et₂O (250 mL) and washed once with satbisulfite (40 mL), once with sat NaHCO₃ (40 mL), once with brine (40 mL)and dried over anhydrous MgSO₄. Purification by flash chromatography onsilica gel using gradient elution with hexanes/ethyl acetate (10:1-7:1)gave 2.22 g (64%) of the iodide 17a as an amber oil.

EXAMPLE 9

Reduction of the alkynyl iodide:

BH₃.DMS (0.846 mL, 8.92 mmol) was added to a solution of cyclohexene(1.47 mL, 17.9 mmol) in Et₂O (60 mL) at 0° C. The reaction was thenwarmed to rt After 1 h, the iodide x (2.22 g, 5.95 mmol) was added toEt₂O. After 3 h, AcOH (1.0 mL) was added. After 30 additional min, thesolution was poured into sat NAHCO, and extracted with Et₂O (3×100 mL).The combined organics were then washed once with brine (50 mL) and driedover anhydrous MgSO₄. Purification by flash chromatography on silica geleluting with hexanes/ethyl acetate (6:1) gave 1.45 g (65%) of the vinyliodide 18 as a yellow oil.

EXAMPLE 10

MOM removal:

To a solution of iodide 18 (1.45 g, 3.86 mmol) in CH₂Cl₂ (40 mL) at rtwas added thiophenol (1.98 mL, 19.3 mmol) and BF₃.Et₂O (1.90 mL, 15.43mmol). After 22 h, the reaction was poured into EtOAc (150 mL) andwashed with 1N NaOH (2×50 mL) and dried over anhydrous MgSO₄.Purification by flash chromatography on silica gel using gradientelution with hexanes/ethyl acetate (4:1-2:1-1:1) gave 1.075 g (86%) ofthe alcohol 18a as a pale yellow oil.

EXAMPLE 11

Acetate formation:

To a solution of alcohol 18a (1.04 g, 3.15 mmol) in CH₂Cl₂ (30 mL) wasadded pyridine (2.52 mL, 25.4 mmol), acetic anhydride (1.19 mL, 12.61mmol) and DMAP (0.005 g). After 1 h, the volatiles were removed invacuo. Purification of the resulting residue by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (7:1) gave 1.16 g (99%) ofthe acetate 19 as a pale yellow oil. IR(film):1737, 1368, 1232, 1018cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) d 6.97 (s, 1H), 6.53 (s, 1H), 6.34 (dd,J=17.5, 1.0 Hz, 1H), 6.18 (dd, J=13.7, 6.9 Hz, 1h), 5.40 (t, J=6.4 Hz,1H), 2.70 (s, 3h), 2.61 (m, 2H), 2.08 (2s, 6H). ¹³C NMR (CDCl₃, 125 MHz)d 169.8, 164.4, 152.2, 136.4, 136.1, 120.6, 116.4, 85.1, 38.3, 21.0,19.1, 14.7; [α]_(D)=−28.8 (c=1.47, CHCl₃).

EXAMPLE 12

To a solution of alcohol 4 (2.34 g, 3.62 mmol) and 2,6-lutidine (1.26mL, 10.86 mmol) in CH₂ _(Cl) ₂ (23 mL) at 0° C. was treated with TBSOTf(1.0 mL, 4.34 mmol). After stirring for 1.5 h at 0° C. the reactionmixture was quenched with MeOH (200 uL) and the mixture stirred anadditional 5 min. The reaction mixture was diluted with Et₂O (100 mL)and washed successively with 1 N HCl (25 mL), water (25 mL), and brine(25 mL). The solution was dried over MgSO₄, filtered, and concentrated.The residue was purified by flash chromatography on silica gel elutingwith 5% Et₂O in hexanes to provide compund 7 (2.70 g, 98%) as acolorless foam.

EXAMPLE 13

A solution of compound 7 (2.93 g, 3.85 mmol) in CH₂Cl₂/H₂O (20:1, 80 mL)was treated with DDQ (5.23 g, 23.07 mmol) and the resulting suspensionwas stirred at room temperature for 24 h. The reaction mixture wasdiluted with Et₂O (200 mL) and washed with aqueous NaHCO₃ (2×40 mL). Theaqueous layer was extracted with Et₂O (3×40 mL) and the combined organicfractions were washed with brine (50 mL), dried over MgSO₄, filtered,and concentrated. Purification of the crude oil by flash chromatographyon silica gel eluting with 30% ether in hexanes afforded alcohol 7A(2.30 g, 89%) as a colorless oil: IR (film) 3488, 1471, 1428, 1115, 1054cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) d 7.70 (6 H, dd, J=8.0, 1.5 Hz), 7.44 (9H, s), 4.57 (1 H, d, J=3.5 Hz), 4.19 (1 H, s), 3.67 (1 H, d, J=8.5 Hz),3.06 (1 H, dd, J=11.5, 5.0 Hz), 2.89 (1 H, dd, J=11.5, 5.0 Hz), 2.68 (1H, d, J=13.5 Hz), 2.59 (1 H, d, J=13.5 Hz), 2.34 (1 H, dt, J=12.0, 2.5Hz), 2.11 (1 H, m), 1.84 (1 H, dt, J=12.0, 2.5 Hz), 1.76 (2 H, m), 1.59(2 H, m), 1.34 (3 H, s), 1.13 (3 H, d, J=7.5 Hz), 1.10 (3 H, s), 0.87 (9H, s), 0.84 (3 H, d, J=12.0 Hz), 0.02 (3 H, s), 0.01 (3 H, s); ¹³C NMR(CDCl₃, 125 MHz) d 136.18, 134.66, 130.16, 127.84, 78.41, 75.91, 63.65,59.69, 45.43, 45.09, 37.72, 30.84, 30.50, 26.23, 25.89, 22.42, 21.05,18.40, 15.60, 14.41, −3.23, −3.51; [α]_(D)=−0.95 (c=0.173, CHCl₃).

EXAMPLE 14

To a solution of oxalyl chloride (414 μL, 4.74 mmol) in CH₂Cl₂ (40 mL)at −78° C. was added dropwise DMSO (448 uL, 6.32 mmol) and the resultingsolution was stirred at −78° C. for 30 min. Alcohol 7a (2.12 g, 3.16mmol) in CH₂Cl₂ (20 mL) was added and the resulting white suspension wasstirred at −78° C. for 45 min. The reaction mixture was quenched withEt₃N (2.2 mL, 15.8 mmol) and the solution was allowed to warm to 0° C.and stirred at this temperature for 30 min. The reaction mixture wasdiluted with Et₂O (100 mL) and washed successively with aqueous NH₄Cl(20 mL), water (20 mL), and brine (20 mL). The crude aldehyde waspurified by flash chromatography on silica gel eluting with 5% Et₂O inhexanes to provide aldehyde 8 (1.90 g, 90%) as a colorless oil.

EXAMPLE 15

A solution of (methoxymethyl)triphenylphosphonium chloride (2.97 g, 8.55mmol) in THF (25 mL) at 0° C. was treated with KO′Bu (8.21 mL, 1M inTHF, 8.1 mmol). The mixture was stirred at 0° C. for 30 min. Aldehyde 8(3.1 g, 4.07 mmol) in THF (10 mL) was added and the resulting solutionwas allowed to warm to room temperature and stirred at this temperaturefor 2 h. The reaction mixture was quenched with aqueous NH₄Cl (40 mL)and the resulting solution extracted with Et₂O (3×30 mL). The combinedEt₂O fractions were washed with brine (20 ml), dried over MgSO₄,filtered, and concentrated. The residue was purified by flashchromatography on silica gel eluting with 5% Et₂O in hexanes to yieldcompound 9 (2.83 g, 86%) as a colorless foam.

EXAMPLE 16

To a solution of compound 9 (2.83 g, 3.50 mmol) in dioxane/H₂O (9:1, 28mL) was added pTSA.H₂O (1.0 g, 5.30 mmol) and the resulting mixture washeated to 50° C. for 2 h. After cooling to room temperature the mixturewas diluted with Et₂O (50 mL) and washed with aqueous NaHCO₃ (15 mL),brine (20 ml), dried over MgSO₄, filtered, and concentrated to providealdehyde 9a (2.75 g, 99%) as a colorless foam.

EXAMPLE 17

Methyltriphenylphosphonium bromide (1.98 g, 5.54 mmol) in THF (50 mL) at0° C. was treated with lithium bis(trimethylsilyl)amide (5.04 mL, 1M inTHF, 5.04 mmol) and the resulting solution was stirred at 0° C. for 30min. Aldehyde 9a (2.0 g, 2.52 mmol) in THF (5.0 mL) was added and themixture was allowed to warm to room temperature and stirred at thistemperature for 1 h. The reaction mixture was quenched with aqueousNH₄Cl (15 mL) and extracted with Et₂O (3×20 mL). The combined Et₂Ofractions were washed with brine (15 mL), dried over MgSO₄, filtered,and concentrated. The residue was purified by flash chromatography onsilica gel eluting with 5% Et₂O in hexanes to afford compound 10 (1.42g, 76%) as a colorless foam.

EXAMPLE 18

A solution of compound 10 (1.0 g, 1.34 mmol) in MeOH/THF (2:1, 13 mL)was treated with [bis(trifluoroacetoxy)iodobenzene] (865 mg, 2.01 mmol)at room temperature. After 15 min the reaction mixture was quenched withaqueous NaHCO₃ (25 mL). The mixture was extracted with Et₂O (3×25 mL)and the combined Et₂O fractions were washed with brine, dried overMgSO₄, filtered, and concentrated. Purification of the residue by flashchromatography on silica gel eluting with 5% Et₂O in hexanes providedcompound 11 (865 mg, 92%) as a colorless foam: IR (film) 1428, 1252,1114, 1075, 1046 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) d 7.61 (6 H, dd, J=7.9,1.4 Hz), 7.38 (9 H, s), 5.47 (1 H, m), 4.87 (1 H, d, J=10.0 Hz), 4.76 (1H, d, J=15.9 Hz), 4.30 (1 H, d, J=3.7 Hz), 3.95 (1 H, s), 3.56 (1 H, dd,J=7.5, 1.4 Hz), 3.39 (3 H, s), 2.84 (3 H, s), 2.02 (1 H, m), 1.64 (2 H,m), 1.34 (1 H, m), 1.11 (3 H, s), 1.02 (3 H, d, J=7.4 Hz), 0.90 (3 H,s), 0.85 (9 H, s), 0.62 (3 H, d, J=6.8 Hz), −0.04 (3 H, s), −0.05 (3 H,s); ¹³C NMR (CDCl₃, 125 MHz) d 138.29, 135.79, 135.04, 129.86, 127.78,114.98, 110.49, 60.11, 55.57, 46.47, 43.91, 36.82, 34.21, 26.26, 19.60,18.60, 17.08, 16.16, 13.92, −2.96, −3.84; [α]_(D)=+1.74 (c=0.77, CHCl₃).

EXAMPLE 19

Suzuki Coupling:

To a solution of olefin 11 (0.680 g, 1.07 mmol) in THF (8.0 ml) wasadded 9-BBN (0.5 M soln in THF, 2.99 mL, 1.50 mmol). In a separateflask, the iodide 19 (0.478 g, 1.284 mmol) was dissolved in DMF (10.0mL). CsCO₃ (0.696 g, 2.14 mmol) was then added with vigorous stirringfollowed by sequential addition of Ph₃As (0.034 g, 0.111 mmol),PdCl₂(dppf)₂ (0.091 g, 0.111 mmol) and H₂O (0.693 mL, 38.5 mmol). After4 h, then borane solution was added to the iodide mixture in DMF. Thereaction quickly turned dark brown in color and slowly became paleyellow after 2 h. The reaction was then poured into H₂O (100 mL) andextracted with Et₂O (3×50 mL). The combined organics were washed withH₂O (2×50 mL), once with brine (50 mL) and dried over anhydrous MgSO₄.Purification by flash chromatography on silica gel eluting withhexanes/ethyl acetate (7:1) gave 0.630 g (75%) of the coupled product 20as a pale yellow oil.

EXAMPLE 20

Hydrolysis of dimethyl acetal 21:

The acetate 20 (0.610 g, 0.770 mmol) was dissolved in dioxane/H₂O (9:1,15 mL) and p-TSAH 20 (0.442 g, 2.32 mmol) was added. The mixture wasthen heated to 55° C. After 3 h, the mixture was cooled to rt and pouredinto Et₂O. This solution was washed once with sat NaHCO₃ (30 mL), oncewith brine (30 mL) and dried over anhydrous MgSO₄. Purification by flashchromatography on silica gel eluting with hexanes/ethyl acetate (7:1)gave 0.486 g (85%) of the aldehyde 21 as a pale yellow oil. IR (film)1737, 1429, 1237, 1115, 1053 cm⁻¹; ¹H NMR (CDCl₃, 500 MHz) d 9.74 (1 H,s), 7.61 (6 H, dd, J=7.8, 1.2 Hz), 7.38 (9 H, m), 6.94 (1 H, s), 6.53 (1H, s), 5.39 (1 H, m), 5.31 (1 H, m), 5.29 (1 H, t, J=6.9 Hz), 4.61 (1 H,d, J=4.3 Hz), 3.50 (1 H, dd, J=5.2, 2.6 Hz), 2.70 (3 H, s), 2.48 (2 H,m), 2.14 (1 H, m), 2.09 (3 H, s), 2.07 (3 H, s), 1.83 (2 H, m), 1.41 (1H, m), 1.18 (1 H, m), 1.01 (3 H, s), 0.99 (3 H, s), 0.91 (3 H, d, J=7.4Hz), 0.85 (9 H, s), 0.69 (1 H, m), 0.58 (3 H, d, J=6.8 Hz), −0.05 (3 H,s), −0.06 (3 H, s); ¹³C NMR (CDCl₃, 125 MHz) d 205.46, 170.01, 164.49,152.46, 137.10, 135.60, 134.22, 132.55, 130.65, 127.84, 123.82, 120.66,116.19, 81.09, 78.47, 76.73, 51.66, 43.14, 38.98, 30.99, 30.42, 27.63,26.10, 21.15, 20.92, 20.05, 19.15, 18.49, 15.12, 14.70, 12.75, −3.25,|31 4.08; [α] _(D)=−18.7 (c=0.53, CHCl₃).

EXAMPLE 21

Aldol:

To a solution of the acetate-aldehyde 21 (84 mg, 0.099 mmol) in THF at−78° C. was added KHMDS (0.5M in toluene, 1.0 ml, 0.5 mmol)) dropwise.The resulting solution was stirred at −78° C. for 30 min. Then thereaction mixture was cannulated to a short pad of silica gel and washedwith ether. The residue was purified by flash chromatography (silica,12% EtOAc in hexane) to give the lactone 22 (37 mg of 3-S and 6 mg of3-R, 51%) as white foam.

EXAMPLE 22

Monodeprotection:

Lactone 22 (32 mg, 0.0376 mmol) was treated with 1 ml of pyridinebuffered HF-pyridine-THF solution at room temperture for 2 h. Thereaction mixture was poured into saturated aqueous NaHCO₃ and extractedwith ether. The organic layer was washed in sequence with saturatedCuSO₄(10 ml×3) and saturated NaHCO₃ (10 ml), then dried over Na₂SO₄ andconcentrated under vacuum. The residue was purified by flashchromatography (silica, 25% EtOAc in hexane) and to give diol 22a (22mg, 99%) as white foam.

EXAMPLE 23

TBS-protection: To a cooled (−30° C.) solution of diol 22a (29 mg,0.0489 mmol) and 2,6-lutidine (0.017 ml, 0.147 mmol) in anhydrous CH₂Cl₂(1 ml) was added TBSOTf (0.015 ml, 0.0646 mmol). The resulting solutionwas then stirred at −30° C. for 30 min. The reaction was quenched with0.5M HCl (10 ml) and extracted with ether (15 ml). Ether layer waswashed with saturated NaHCO₃, dried (Na₂SO₄, and concentrated in vacuo.Purifiction of the residue by flash chromatogrphy (silica, 8% EtOAc inhexane) afforded TBS ether 22B (32 mg, 93%) as white foam.

EXAMPLE 24

Ketone Formation:

To a solution of alcohol 22B (30 mg, 0.0424 mmol) in CH₂Cl₂ (2.0 mL) at25° C. was added Dess-Martin periodinane (36 mg, 0.0848 mmol) in oneportion. The resulting solution was then allowed to stir at 25° C. for1.5 h. The reaction was quenched by the addition of 1:1 saturatedaqueous sodium bicarbonate:sodium thiosulfate (10 ml) and stirred for 5min. The mixture was then extracted with ether (3×15 ml). The organiclayer was dried (Na₂SO₄), filtered, and concentrated in vacuo.Purification of the residue by flash chromatography (silica, 8% EtOAc inhexane) provided ketone 22C (25 mg, 84%) as white foam. IR(film): 2928,1745, 1692, 1254, 1175, 836 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) d 6.97 (s, 1H),6.57 (s, 1H), 5.53 (dt, J=3.4, 11.1 Hz, 1H), 5.37 (dd, J=16.4, 9.9 Hz,1H), 5.00 (d, J=10.3 Hz, 1H), 4.02 (d, J=9.7 Hz, 1H), 3.89 (d, J=8.7 Hz,1 H), 3.00 (m, 1H), 2.82 (d, J=6.5 Hz, 1H), 2.71 (m, 5H), 2.36 (q,J=10.7 Hz, 1H), 2.12 (, 3H), 2.07 (dd, J=8.2, 1H), 1.87 (bs, 1H), 1.49(m, 3H), 1.19 (m, 5H), 1.14 ts, 3H), 1.08 (d, J=6.8 Hz, 3H), 0.94 (m,12H), 0.84 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.07 (s, 3H), −0.098 (s,3H); ¹³C NMR (CDCl₃, 125 MHz) d 218.7, 170.1, 164.5, 152.6, 137.9,133.9, 124.8, 119.6, 115.9, 72.7, 53.2, 43.9, 41.0, 40.3, 32.9, 32.3,28.4, 27.1, 26.3, 26.1, 26.0, 19.2, 19.1, 18.3, 18.2, 17.1, 16.0, 15.2,14.3, −4.2, −4.4, −4.6, −4.8 ; [α]_(D)=−21.93 (c=1.4, CHCl₃).

EXAMPLE 25

Desoxy compound:

To a solution of TBS ether 22C (27 mg, 0.038 mmol) in THF(1 ml) at 25°C. in a plastic vial was added dropwise HF-pyridine (0.5 ml). Theresulting solution was allowed to stir at 25° C. for 2 h. The reactionmixture was diluted with chloroform (2 ml) and very slowly added tosatured sodium bicarbonate (20 ml). The mixture was extracted with CHCl₃(20ml×3). The organic layer was dried (Na₂SO₄), filtered, andconcentrated in vacuo. Purification of the residue by flashchromatography (silica, 30% EtOAc in hexane) provided diol 23 (18 mg,99%) as white foam: IR(film): 3493, 2925, 1728, 1689, 1249 cm⁻¹; ¹H NMR(CDCl₃, 500 MHz) d 6.96 (s, 1H), 6.59 (s, 1H), 5.44 (dt, J=4.3, 10.4 Hz,1H), 5.36 (dt, J=5.1, 10.2 Hz, 1H), 5.28 (dd, J=1.7, 9.8 Hz, 1H), 4.11(d, J=7.2 Hz, 1H), 3.74 (s, 1H), 3.20 (d, J=4.5 Hz, 1H), 3.14 (dd,J=2.2, 6.8 Hz, 1H), 3.00 (s, 1H), 2.69 (m, 4H), 2.49 (dd, J=1.3, 15.1Hz, 1H), 2.35 (dd, J=2.5, 15.1 Hz, 1H), 2.27(m, 1H), 2.05 (m, 1H), 2.04(s, 3H), 2.01 (m, 1H) 1.75 (m, 1H), 1.67 (m, 1H), 1.33 (m, 4H), 1.21 (s,1H), 1.19 (m, 2H), 1.08 (d, J=7.0 Hz, 3H), 1.00 (s, 3H), 0.93 (d, J=7.1Hz, 3H); ¹³C NMR (CDCl₃, 125 MHz) d 226.5, 176.5, 171.1, 158.2, 144.7,139.6, 131.1, 125.7, 122.0, 84.6, 80.2, 78.6, 59.4, 47.9, 45.4, 44.6,38.5, 37.9, 33.7, 33.6, 28.7, 25.1, 25.0, 21.9, 21.7, 19.6;[α]_(D)=−84.7 (c=0.85, CHCl₃).

EXAMPLE 26

Epothilone:

To a cooled (−50° C.) solution of desoxyepothilone (9 mg, 0.0189 mmol)in dry CH₂Cl₂ (1 ml) was added freshly prepared dimethyldioxirane (0.95ml, 0.1 M in acetone). The resulting solution was allowed to warm up to−30° C. for 2 h. A stream of nitrogen was then bubbled through thesolution to remove excess DMDO. The residue was purified by flashchromatography (silica, 40% EtOAc in hexane) and afforded epothilone A(4.6 mg, 49%) as colorless solid and 0.1 mg of cisepoxide diastereomer.This material was identical with the natural epothilone A in allrespects.

EXAMPLE 27

Procedure for Ring-closing Olefin Metathesis:

To a stirred solution of diene 24 (5 mg, 0.0068 mmol) in dry benzene(1.5 mL) was added Grubbs's catalyst (2.8 mg, 0.0034 mmol). After 12 h,an additional portion of catalyst was added (2.8 mg). After anadditional 5 h, the reaction was concentrated. Purification by silicagel chromatography eluting with hexanes/ethyl acetate (11:1) gave thelactone 23 (3.5 mg, 94%, 2:1 E/Z).

EXAMPLE 28

Preparation of Compound 19:

Alcohol 2A:

A mixture of (S)-(−)-1,1¹-bi-2-naphthol (259 mg. 0.91 mmol), Ti(O-i-Pr)₄(261 μL;0.90 mmol), and 4 Å sieves (3.23 g) in CH₂Cl₂ (16 mL) was heatedat reflux for 1 h. The mixture was cooled to rt and aldehyde 1 wasadded. After 10 min. the suspension was cooled to −78° C., and allyltributyltin (3.6 mL; 11.60 mmol) was added. The reaction mixture wasstirred for 10 min at −78° C. and then placed in a −20° C. freezer for70 h. Saturated NaHCO₃ (2 mL) was added, and the mixture was stirred for1 h, poured over Na₂SO₄, and then filtered through a pad of MgSO₄ andcelite. The crude material was purified by flash chromatography(hexanes/ethyl acetate, 1:1) to give alcohol 2A as a yellow oil (1.11 g;60%).

EXAMPLE 29

Acetate 3A:

To a solution of alcohol 2A (264 mg; 1.26 mmol) in CH₂Cl₂ (12 mL) wasadded DMAP (15 mg: 0.098 mmol), Et₃N (0.45 mL; 3.22 mmol), and Ac₂O(0.18 mL; 1.90 mmol). After 2 h, the reaction mixture was quenched by 20mL of H₂O, and extracted with EtOAC (4×20 ml). The combined organiclayer was dried with MgSO₄, filtered, and concentrated. Flashchromatrography (EtOAC/hexanes, 1:3) afforded acetate 3A as a yellow oil(302 mg; 96%).

EXAMPLE 30

Vinyl Iodide 19:

To a solution of acetate 3A (99 mg; 0.39 mmol) in acetone at 0° C. wasadded H₂O (4 drops), OsO₄ (2.5% wt. in butyl alcohol; 175 μL; 0.018mmol), and N-methyl-morpholine-N-oxide (69 mg; 0.59 mmol). The mixturewas stirred at 0° C. for 2 h and 45 min and then quenched with Na₂SO₃.The solution was poured to 10 mL of H₂O and extracted with EtOAc (5×10mL). The combined organic layer was dried over MgSO4, filtered, andconcentrated.

To a solution of this crude product in THF/H₂O (4 mL, 3:1) was addedNaIO₄ (260 mg; 1.22 mmol). After 1.25 h, the reaction mixture was thenquenched with 10 mL of H₂O and concentrated. The residue was extractedwith EtOAc (5×10 mL). The organic layer was dried over MgSO₄ filtered,and concentrated. Flash chromatography (EtOAc/hexanes, 1:1) gave ayellow oil (80 mg) which contained unidentified by-product(s). Thismixture was used without further purification.

To a solution of (Ph₃P ⁺CH₂I)I⁻ (100 mg; 0.19 mmol) in 0.25 mL of THF atrt was added 0.15 mL (0.15 mmol) of NaHMDS (1M in THF). To the resultingsolution at −78° C. was added HMPA (22 μL; 0.13 mmol) and the productfrom previous step (16 mg) in THF (0.25 mL). The reaction mixture wasthen stirred at rt for 30 min. After the addition of hexanes (10 mL),the solution was extracted with EtOAc (4×10 mL). The combined EtOAClayer was dried (MgSO₄), filtered, and concentrated. Preparative TLC(EtOAc/hexanes, 2.3) afforded vinyl iodide 19 as a yellow oil (14 mg;50% for three steps).

EXAMPLE 31

Iodoolefin acetate 8C:

To a suspension of ethyltriphenylphosphonium iodide (1.125 g, 2.69 mmol)in THF (10 mL) was added nBuLi (2.5 M soln in hexanes, 1.05 mL, 2.62mmol) at rt. After disappearance of the solid material, the solution wasadded to a mixture of iodine (0.613 g, 2.41 mmol) in THF (20 mL) at −78°C. The resulting suspension was vigorously stirred for 5 min at −78° C.,then warmed up −20° C., and treated with sodium hexamethyldisilazane (1M soln in THF, 2.4 mL, 2.4 mmol). The resulting red solution was stirredfor 5 min followed by the slow addition of aldehyde 9C (0.339 g, 1.34mmol). The mixture was stirred at −20° C. for 40 min, diluted withpentane (50 mL), filtered through a pad of celite, and concentrated.Purification of the residue by flash column chromatography(hexanes/ethyl acetate, 85:15) gave 0.202 g (25% overall from vinylacetate 10C) of the vinyl iodide 8C as a yellow oil. IR (film): 2920,1738, 1234 cm⁻¹; ¹H NMR (CDCl₃): δ6.98 (s, 1H), 6.56 (s, 1H), 5.42 (dd,J=5.43, 6.57 Hz, 1H), 5.35 (t, J=6.6 Hz, 1H), 2.71 (s, 3H), 2.54 (q,J=6.33, 2H), 2.50 (s, 3H), 2.09 (s, 6H); ¹³C NMR (CDCl₃): δ170.1, 164.6,152.4, 136.9, 130.2, 120.6, 116.4, 103.6, 40.3, 33.7, 21.2, 19.2, 14.9;[α]_(D)=−20.7° (c=2.45, CHCl₃).

EXAMPLE 32

Acetal 13C:

To a solution of olefin “7C” (0.082 g, 0.13 mmol) in THF (0.5 ml) wasadded 9-BBN (0.5 M soln in THF, 0.4 mL, 0.2 mmol). After stirring at rt.for 3.5 h, an additional portion of 9-BBN (0.5 M soln in THF, 0.26 mL,0.13 mmol) was added. In a separate flask, iodide 8C (0.063 g, 0.1 6mmol) was dissolved in DMF (0.5 mL). Cs₂CO₃ (0.097 g, 0.30 mmol) wasthen added with vigorous stirring followed by sequential addition ofPdCl₂(dppf)₂ (0.018 g, 0.022 mmol), Ph₃As (0.0059 g, 0.019 mmol), andH₂O (0.035 mL, 1.94 mmol). After 6 h, then borane solution was added tothe iodide mixture in DMF. The reaction quickly turned dark brown incolor and slowly became pale yellow after 3 h. The reaction was thenpoured into H₂O (10 mL) and extracted with Et₂O (3×15 mL). The combinedorganic layers were washed with H₂O (3×15 mL), brine (1×20 mL), driedover MgSO₄, filtered, and concentrated. Flash column chromatography(hexanes/ethyl acetate, 9:1) gave 0.089 g (77%) of the coupled product13C as a yellow oil.

EXAMPLE 33

Aldehyde 14C: Acetal 13C (0.069 & 0.077 mmol) was dissolved indioxane/H₂O (9:1, 1 mL) and pTSA-H₂O (0.045 g, 0.237 mmol) was added.The mixture was then heated to 55° C. After 3 h, the mixture was cooledto rt, poured into Et₂O, and extracted with Et₂O (4×15 mL). The combinedether solutions were washed with sat NaHCO₃ (1×30 mL), brine (1×30 mL),dried over MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 3:1) gave 0.046 g (71%) of thealdehyde 14C as a pale yellow oil.

EXAMPLE 34

Macrocycle 15C4SR):

To a solution of aldehyde 14C (0.021 g, 0.024 mmol) in THF (5 mL) at−78° C. was added KHMOS (0.5 M soln in toluene, 0.145 mL, 0.073 mmol).The solution was stirred at −78° C. for 1 h, then quenched with sat'dNH₄Cl, and extracted with ether (3×15 mL). The combined organic layerswere dried with MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 7:1) gave 0.008 g of the desiredα-alcohol 15C-(S) and 0.006 g of β-alcohol 15C-(R) (67% total) as paleyellow oils.

EXAMPLE 35

Macrocycle 15C-(S):

To a solution of β-alcohol 15C-(R) (0.006 g, 0.0070 mmol) in 0.5 mL ofCH₂Cl₂ at rt. was added Dess-Martin periodinane (0.028 g, 0.066 mmol).After 0.5 h, an additional portion of Dess-Martin periodinane (0.025 mg,0.059 mmol) was added. The resulting solution was stirred at rt foradditional 1 h, then treated with ether (2 mL) and sat'd Na₂S₂O₃/sat'dNaHCO₃ (3 mL, 1:1), poured into H₂O (20 mL), and extracted with ether(4×10 mL). The combined ether solutions were washed with H₂O (1×30 mL),brine (1×30 mL), dried with MgSO₄, filtered, and concentrated. To asolution of crude ketone 15C′ in MeOH/THF (2 mL, 1:1) at −78° C. wasadded NaBH₄ (0.015 g, 0:395 mmol). The resulting solution was stirred atrt for 1 h, quenched with sat NH₄Cl, and extracted with ether (3×15 mL).The organic layers were dried with MgSO₄, filtered, and concentrated.Flash column chromatography (hexanes/ethyl acetate, 9:1) gave 0.0040 g(67%) of the α-alcohol 15C-(S) as a pale yellow oil and 0.0006 g ofβ-alcohol 15C-(R).

EXAMPLE 36

Diol 15C″:

The silyl ether 15C-(S) (0.010 g, 0.012 mmol) was dissolved inHF-pyridine/pyridine/THF (1 mL). The solution was stirred at rt. for 2h, then diluted with Et₂O (1 mL), poured into a mixture of Et₂O/sat.NaHCO₃ (20 mL, 1:1), and extracted with Et₂O (4×10 mL). The Et₂Osolutions were washed with sat CuSO₄ (3×30 ml), sat NaHCO₃ (1×30 mL),brine (1×30 mL), dried with MgSO₄, filtered, and concentrated. Flashcolumn chromatography (hexanes/ethyl acetate, 9:1) gave 0.0066 g (93%)of the diol 15C″ as a pale yellow oil.

EXAMPLE 37

Alcohol 15C′″:

To a solution of diol 15C″ (0.0066 g, 0.011 mmol) in 0.5 mL of CH₂Cl₂ at78° C. was added 2,6-lutidine (7 μL, 0.060 mmol) and TBSOTf (5 μL, 0.022mmol). The resulting solution was stirred at −30° C. for 0.5 h, thenquenched with H₂O (5 mL), and extracted with Et₂O (4×10 mL). Theethersolutions were washed with 0.5 M HCl (1×10 mL), sat'd NaHCO₃ (1×10mL), dried over MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 93:7) gave 0.0070 g (89%) of thealcohol 15C′″ as a pale yellow oil.

EXAMPLE 38

Ketone 16C:

To a solution of alcohol 15C′″ (0.006 g, 0.0083 mmol) in 0.5 mL ofCH₂Cl₂ at rt. was added Dess-Martin periodinane (0.030 g, 0.071 mmol).After 1.25 h, another portion of Dess-Martin periodinane (0.025 mg,0.059 mmol) was added. The resulting solution was stirred at rt foradditional 0.75 h, treated with ether (1 mL) and sat'd Na₂S₂O₃/sat'dNaHCO₃ (2 mL, 1:1), poured into H₂O (20 mL), and extracted with ether(4×10 mL). The ether solution was washed with sat NaHCO₃ (1×20 mL),dried with MgSO₄, filtered, and concentrated. Flash columnchromatography (hexanes/ethyl acetate, 9:1) gave 0.0040 g (67%) of theketone 16C as a pale yellow oil.

EXAMPLE 39

Desoxyepothiolone B (2C):

To a solution of ketone 16C (0.004 g, 0.0056 mmol) in THF (0.35 mL) wasadded HF-pyridine (0.25 mL) dropwise over 20 min. The solution wasstirred at rt for 1.5 h, diluted with CHCl₃ (2 mL), poured into sat'dNaHCO₃/CHCl₃ (20 mL, 1:1) slowly, and extracted with CHCl₃ (4×10 mL).The combined CHCl₃ layers were dried with MgSO₄, filtered, andconcentrated. Flash column chromatography (hexanes/ethyl acetate, 3:1)gave 0.0022 g (80%) of the desoxyepothilone B 2C as a pale yellow oil.

EXAMPLE 40

Epothilone B (2):

To a solution of desoxyepothilone B (0.0022 g, 0.0041 mmol) in CH₂Cl₂(0.25 ml) at −50° C. was added dimethyldioxirane (0.1 mL, 0.0095 mmol)dropwise. The resulting solution was stirred at −50° C. for 1 h. Thedimethyldioxirane and solvent were removed by a stream of N₂. Theresidue was purified by flash column chromatography (hexanes/ethylacetate, 1:1) gave 0.0015 g (70%) of epothiolone B (2) as a pale yellowoil which was identical with an authentic sample in ¹H NMR, IR, massspectrum, and [α]_(D).

EXAMPLE 41

8-Desmethylepothilone A

Crotylation product:

To a stirred mixture of potassium tert-butoxide (1.0 M soln in THF, 50.4mL, 50.4 mmol), THF (14 mL), and cis-2-butene (9.0 mL, 101 mmol) at −78°C. was added n-BuLi (1.6 M, in hexanes, 31.5 mL, 50.4 mmol). Aftercomplete addition of n-BuLi, the mixture was stirred at −45° C. for 10min and then cooled to −78° C. (+)-B-Methoxydiisopinocampheylborane(19.21 g, 60.74 mmol) was then added dropwise in Et₂O (10 mL). After 30min, BF₃.Et₂O (7.47 mL, 60.74 mmol) was added followed by aldehyde 4D(9.84 g, 60.74 mmol) in THF (15 mL) generating a viscous solution whichcould not be stirred. The mixture was shaken vigorously every 10 min toensure homogeneity. After 3 h at −78° C., the reaction was treated with3N NaOH (36.6 mL, 110 mmol) and 30% H₂O₂ (15 mL) and the solutionbrought to reflux for 1 h. The reaction was poured into Et₂O (300 mL)and washed with H₂O (100 mL), brine (30 mL) and dried over anhydrousMgSO₄. The crude material was placed in a bulb-to-bulb distillationapparatus to remove the ligand from the desired product. Heating at 80°C. at 2 mm Hg removed 90% of the lower boiling ligand. Furtherpurification of the alcohol 4D was achieved by flash chromatography onsilica gel eluting with Et₂O in CH₂Cl₂ (2%-4%) to give pure alcohol 4Das a clear oil. The erythro selectivty was >50:1 as judged by ¹H NMRspectroscopy. The product was determined to be 87% ee by formation ofthe Mosher ester: IR (film): 3435, 2861, 1454, 1363, 1099 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ7.34 (5 H, m), 5.80 (1 H, m), 5.09 (1 H, dd, J=1.6,8.3 Hz), 5.04 (1 H, d, J=1.6 Hz), 4.52 (2 H, s), 3.51 (2 H, t, J=5.8Hz), 3.47 (1 H, m), 2.27 (2 H, m), 1.73 (3 H, m), 1.42 (1 H, m), 1.04 (3H, d, J=6.9 Hz); ¹³C NMR (CDCl₃, 100 MHz) δ141.1, 138.2, 128.3, 127.6,127.5, 115.0, 74.5, 72.9, 70.4, 43.7, 31.3, 26.5, 14.6.

EXAMPLE 42

TBS ether 5D:

Alcohol 4D (5.00 g, 21.4 mmol) was dissolved in CH₂Cl₂ (150 mL) and2,6-lutidine (9.97 mL, 85.6 mmol) was added. The mixture was cooled to0° C. and TBSOTf (9.83 mL, 42.8 mmol) was slowly added. The reaction wasthen warmed to rt. After 1 h, the reaction was poured into Et₂O (300 mL)and washed once with 1 N HCl (50 mL), once with sat NaHCO₃ (50 mL), oncewith brine (30 mL) and dried over anhydrous MgSO₄. Purification by flashchromatography on silica gel eluting with hexanes/diethyl ether (97:3)gave 6.33 g (85%) of pure olefin 5D as a clear oil: IR (film): 1472,1361, 1255, 1097, 1068 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ7.30 (5 H, m),5.81 (1 H, m), 4.97 (1 H, dd, J=1.4, 4.8 Hz), 4.94 (1 H, d, J=1.1 Hz),3.51 (1 H, q, J=5.1 Hz), 3.41 (2 H, dt, J=2.1, 6.6 Hz), 2.27 (1 H, q,J=5.5 Hz), 1.68 (1 h, m), 1.55 (1 H, m), 1.41 (2 H, m), 0.93 (3 H, d,J=6.9 Hz), 0.85 (9 H, s), −0.01 (6 H, s); ¹³C NMR (CDC₃, 100 MHz)δ141.2, 138.6, 128.3, 127.6, 127.4, 113.9, 75.6, 72.7, 70.6, 42.7, 30.1,25.9, 25.4, 18.1, 15.1, −4.3, −4.4.

EXAMPLE 43

Aldehyde 60:

The olefin 5 (4.00 g, 11.49 mmol) was dissolved in 1:1 MeOH/CH₂Cl₂ (100mL. Pyridine (4.0 mL) was then added and the mixture cooled to −78° C.Ozone was then bubbled through the reaction for 10 minutes before thecolor turned light blue in color. Oxygen was then bubbled through thereaction for 10 min. Dimethyl sulfide (4.0 mL) was then added and thereaction slowly warmed to rt. The reaction was stirred overnight andthen the volatiles were removed in vacuo. Purification by flashchromatography on silica gel eluting with hexanes/ethyl acetate (9:1)gave 3.31 g (82%) of the aldehyde 6D as a clear oil: IR (film): 2856,1727, 1475, 1361, 1253, 1102 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ9.76 (1 H,s), 7.33 (5 H, m), 4.50 (2 H, s), 4.11 (1 H, m), 3.47 (2 H, m), 2.46 (1H, m), 1.50-1.70 (4 H, band), 1.05 (1 H, d, J=7.0 Hz), 0.86 (9 H, s),0.06 (3 H, s), 0.03 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ204.8, 138.3,128.2, 127.4, 127.3, 72.7, 71.7, 69.9, 51.1, 31.1, 25.9, 25.6, 17.8,7.5, −4.4, −4.8.

EXAMPLE 44

Dianion addition product 7D:

The tertbutyl isobutyrylacetate (0.653 g, 3.51 mmol) was added to asuspension of NaH (60% in mineral oil, 0.188 g, 4.69 mmol) in THF (50mL) at rt. After 10 min, the mixture was cooled to 0° C. After anadditional 10 min, n-BuLi (1.6 M in hexanes, 2.20 mL, 3.52 mmol) wasslowly added. After 30 min, the aldehyde 6D (1.03 g, 2.93 mmol) wasadded neat. After 10 min, the reaction was quenched with H₂O (10 mL) andextracted with Et₂O (2×75 mL). The combined organics were washed oncewith brine (30 mL) and dried over anhydrous MgSO₄. The crude reactionmixture contained a 15:1 ratio of diastereomers at C5. Purification byflash chromatography on silica gel eluting with hexanes/ethyl acetate(9:1-7:1) gave 0.723 g (47%) of the desired alcohol 7D as a clear oil:IR (film): 3531, 2953, 1739, 1702, 1367, 1255, 1153 cm⁻¹; ¹H NMR(CDCl₃,400 MHz) δ7.33 (5 H, m), 4.49 (2 H, s), 3.75 (1 H, d, J=2.6 Hz), 3.71 (1H, m), 3.62 (1 H, d, J=16.0 Hz), 3.53 (1 H, d, J=16.0 Hz), 3.44 (2 H, t,J=5.1 Hz), 2.70 (1 H, d, J=2.6 Hz), 1.83 (1 H, m), 1.55 (4 H, m), 1.46(9H, s), 1.17 (3 H, s), 1.11 (3 H, s), 0.89 (9 H, s), 0.82 (3 H, d, J=7.0Hz), 0.09 (6 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ208.9, 167.3, 138.4,128.3, 127.6, 127.5, 81.3, 79.5, 78.7, 72.8, 70.1, 52.4, 47.6, 35.8,30.6, 28.2, 25.9, 25.8, 22.6, 20.5, 17.9, 7.05, −4.0, −4.5.

EXAMPLE 45

Directed reduction:

To a solution of tetramethylammonium triacetoxyborohydride (1.54 g, 5.88mmol) in acetonitrile (4.0 mL) was added anhydrous AcOH (4.0 mL). Themixture was stirred at rt for 30 min before cooling to −10° C. Asolution of the ester 7D (0.200 g, 0.39 mmol) in acetonitrile (1.0 mL)was added to the reaction and it was stirred at −10° C. for 20 h. Thereaction was quenched with 1 N sodium-potassium tartrate (10 mL) andstirred at rt for 10 min. The solution was then poured into sat NaHCO₃(25 ml) and neutralized by the addition of solid Na₂CO₃. The mixture wasthen extracted with EtOAc (3×30 mL) and the organics were washed withbrine (20 mL) and dried over anydrous MgSO₄. Purification by flashchromatography on silica gel eluting with hexanes/ethyl acetate (4:1)gave 0.100 g (50%) of the diol as 10:1 ratio of diastereomeric alcohols.

EXAMPLE 46

Monoprotection of the diol:

The diol (1.76 g, 3.31 mmol) was dissolved in CH₂Cl₂ (100 mL) and cooledto 0° C. 2,6-lutidine (12.2 mL, 9.92 mmol) was added followed by TBSOTf(1.14 mL, 4.96 mmol) and the reaction slowly warmed to rt. After 1 h,the reaction was poured into Et₂O (300 mL) and washed once with 1 N HCl(50 mL), once with sat NaHCO₃ (50 mL), once with brine (30 mL) and driedover anhydrous MgSO₄. Purification by flash chromatography on silica geleluting with hexanes/ethyl acetate (20:1-15:1) gave 2.03 g (95%) of thealcohol 8D as a clear oil, which was used as a mixture of diastereomers.

EXAMPLE 47

C5 Ketone formation:

The alcohol 8D (2.03 g, 3.14 mmol) was dissolved in CH₂Cl₂ (50 mL) andDess-Martin periodinane (2.66 g, 6.28 mmol) was added. After 2 h, a 1:1mixture of sat'd NaHCO/sat Na₂S₂O₃ (20 mL) was added. After 10 min, themixture was poured into Et₂O (300 mL) and the organic layer was washedwith brine (30 ml) and dried over anhydrous MgSO₄. Purification by flashchromatography on silica gel eluting with hexaries/ethyl acetate (15:1)gave 1.85 g (91%) of the ketone (benzyl ether) as a clear oil, which wasused as a mixture of diastereomers.

EXAMPLE 48

Debenzylation:

The ketone (benzyl ether) (1.85 g, 2.87 mmol) was dissolved in EtOH (50mL), and Pd(OH)₂ (0-5 8) was added. The mixture was then stirred underan atmosphere of H₂. After 3 h, the reaction was purged with N₂ and thenfiltered through a pad of celite rinsing with CHCl₃ (100 mL).Purification by flash chromatography on silica gel eluting with ethylacetate in hexanes (12%-15%) gave 1.43 g (90%) of the diastereomericalcohols as a clear oil. The C3 diastereomers were separated by flashchromatography on TLC-grade SiO₂ eluting with ethyl acetate in hexanes(15%):

Alpha isomer: IR (film): 3447, 1732, 1695, 1254, 1156 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ4.24 (1 H, dd, J=3.6, 5.8 Hz), 3.83 (1 H, m), 3.53 (1H, m), 3.06 (1 H, t, J=7.1 Hz), 2.36 (1 H, dd, J=3.6, 17.2 Hz), 2.12 (1H, dd, J=3.9, 17.2 Hz), 1.68 (1 H, t, J=5.4 Hz), 1.54 (2 H, m), 1.41 (1H, m), 1.37 (9 H, s), 1.31 (1 H, m), 1.16 (3 H, s), 1.02 (3 H, s), 0.99(3 H, d, J=6.8 Hz), 0.84 (9 H, s), 0.81 (9 H, s), 0.05 (3 H, s), 0.01 (6H, s), −0.01 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ217.7, 171.3, 80.57,73.5, 73.1, 63.0, 53.4, 26.8, 41.2, 32.1, 28.1, 28.0, 26.0, 25.9, 23.1,19.8, 18.1 (overlapping), 15.3, −4.0, −4.3 (overlapping), −4.8.

Beta isomer: IR (film): 3442, 2857, 1732, 1700, 1472, 1368, 1255 cm⁻¹;¹H NMR (CDCl₃, 400 MHz) δ4.45 (1 H, t, J=5.3 Hz), 3.86 (1 H, m), 3.52 (2H, q, J=5.9 Hz), 3.01 (1 H, m), 2.28 (1 H, dd, J=4.3, 17.1 Hz), 2.16 (1H, dd, J=5.5, 17.1 Hz), 1.67 (1 H, t, J=5.6 Hz), 1.56 (2 H, m), 1.44 (1H, m), 1.37 (9 H, s), 1.34 (1 H, m), 1.13 (3 H, s), 0.97 (3 H, d, J=7.4Hz), 0.96 (3 H, s), 0.83 (9 H, s), 0.79 (9 H, s), 0.01 (3 H, s), 0.00 (6H, s), −0.07 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ217.1, 171.2, 80.6,73.5, 72.1, 62.9, 63.9, 46.4, 41.2, 32.0, 28.1, 28.0, 26.0, 25.9, 21.5;19.5, 18.2, 18.1, 15.8, −4.0, 4.3, −4.4, −4.7.

EXAMPLE 49

Aldehyde formation:

DMSO (0.177 mL, 2.50 mmol) was added to a mixture of oxalyl chloride(0.11 ml, 1.25 mmol) in CH₂Cl₂ (15 mL) at −78° C. After 10 min, thealcohol (0.531 g, 0.96 mmol) was added in CH₂Cl₂ (4 mL). After 20 min,TEA (0.697 mL, 5.00 mmol) was added to the reaction followed by warmingto rt. The reaction was then poured into H₂O (50 mL) and extracted withEt₂O (3×50 mL). The organics were washed once with H₂O (30 mL), oncewith brine (30 mL) and dried over anhydrous MgSO₄. The aldehyde was usedin crude form.

EXAMPLE 50

Wittig olefination to give 9D:

NaHMDS (1.0 M soln in THF, 1.54 mL, 1.54 mmol) was added to a suspensionof methyl triphenylphosphonium bromide (0.690 g, 1.92 mmol) in THF (20mL) at 0° C. After 1 h, the crude aldhyde (0.96 mmol) was added in THF(5 mL). After 15 min at 0° C., H₂O (0.1 mL) was added and the reactionpoured into hexanes (50 mL). This was filtered through a plug of silicagel eluting with hexanes/Et₂O (9:1, 150 mL). The crude olefin 9D wasfurther purified by flash chromatography on silica gel eluting withethyl acetate in hexanes (5%) to give 0.437 g (83% for two steps) of theolefin 9D as a clear oil: IR (film): 2857, 1732, 1695, 1472, 1368, 1255,1156 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ5.72 (1 H, m), 4.91 (2 H, m), 4.25(1 H, dd, J=3.9, 5.4 Hz), 3.81 (1 H, m), 3.05 (1 H, m), 2.38 (1 H, dd,J=7.9, 17.2 Hz), 2.12 (1 H, dd, J=6.6, 17.2 Hz), 2.04 (2 H, q, J=7.5Hz), 1.47 (1 H, m), 1.39 (9 H, s), 1.34 (1 H, m), 1.20 (3 H, s), 1.00 (3H, s), 3.00 (3 H, d, J=6.7 Hz), 0.85 (9 H, s), 0.83 (9 H, s), 0.07 (3 H,s), 0.00 (6 H, s), −0.05 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ217.5,172.1, 137.9, 114.0, 80.4, 74.0, 73.0, 53.0, 46.9, 41.3, 35.1, 29.0,28.1, 26.0, 25.9, 22.8, 20.2, 18.2 (overlapping), 14.9, −4.1, −4.2,−4.3, −4.8.

EXAMPLE 51

TBS ester 10D:

The olefin 9D (0.420 g, 0.76 mmol) was dissolved in CH₂Cl₂ (15 mL) andtreated successively with 2,6-lutidine (1.33 mL, 11.4 mmol) and TBSOTf(1.32 mL, 5.73 mmol). After 7 h, the reaction was poured into Et₂O (100mL) and washed successively with 0.2N HCl (25 mL), brine (20 mL) anddried over anhydrous MgSO₄. The residue was purified by flashchromatography on a short pad of silica gel with fast elution withhexanes/ethyl acetate (20:1) to give the TBS ester 10D as a clear oil.The purification must be done quickly to avoid hydrolysis of the silylester: IR (film): 2930, 1721, 1695, 1472, 1254, 1091 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ5.73 (1 H, m), 4.91 (2 H, m), 4.25 (1 H, dd, J=3.8,5.4 Hz) 3.80 (1 H, q, J=6.8 Hz), 3.06 (1 H, m), 2.50 (1 H, dd, J=3.7,17.3 Hz), 2.19 (1 H, dd, J=5.7, 17.3 Hz), 2.04 (2 H, dd, J=7.6, 15.3Hz), 1.49 (1 H, m), 1.36(1 H, m), 1.21 (3 H, s), 1.00 (3 H, d, J=6.8Hz), 0.88 (9 H, s), 0.85 (9 H, s), 0.83 (9 H, s), 0.22 (3 H, s), 0.22 (3H, s), 0.21 (3 H, 5), 0.06 (3 H, s), 0.01 (6 H, s), −0.05 (3 H, s); ¹³CNMR (CDCl₃, 100 MHz) δ217.3, 172.3, 138.5, 114.4, 74.5, 73.0, 53.2,46.9, 41.8, 35.1, 29.0, 26.0, 225.7, 25.5, 22.8, 20.4, 18.2, 18.1, 17.5,14.9, −2.9, −4.0, −4.2, −4.3, −4.8, −4.9.

EXAMPLE 52

Suzuki coupling:

The acetate acid 13D was purified by flash chromatography on silica geleluting with hexanes/ethyl acetate (7:1-4:1). This was further purifiedby preparative-TLC eluting with hexanes/ethyl acetate (2:1) to removeunreacted vinyl iodide 12D from the acetate acid 13D. Isolated yield ofthe acid was 0.297 g (62% based on 90% purity with borane residues).

EXAMPLE 53

Hydrolysis of acetate acid 13D:

The acetate 13D (0.220 g, 0.297 mmol) was dissolved in MeOH/H₂O (2:1, 15mL) and K₂CO₃ (0.300 g) was added. After 3 h, the reaction was dilutedwith sat NH₄Cl (20 mL) and extracted with CHCl₃ (5×20 mL. Thehydroxy-acid 14D was purified by flash chromatography on silica geleluting with hexanes/ethyl acetate (4:1-2:1) to give 0.146 g (70%) ofthe pure hydroxy acid 14D. IR (film): 3510-2400, 1712, 1694, 1471, 1254,1093 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ6.96 (1 H, s), 6.66 (1 H, s), 5.55(1 H, m), 5.38 (1 H, m), 4.38 (1 H, dd, J=3.4, 6.1 Hz), 4.19 (1 H, t,J=7.5 Hz), 3.84 (1 H, m), 3.05 (1 H, t, J=7.0 Hz), 2.72 (3 H, s), 2.49(1 H, dd, J=3.2, 16.4 Hz), 2.42 (2 H, m), 2.33 (1 H, dd, J=6.2, 16.4Hz), 2.07 (2 H, m), 2.02 (3 H, s), 1.33 (4 H, m), 1.19 (3 H, s), 1.14 (3H, s), 1.06 (3 H, d, J=6.7 Hz), 0.89 (9 H, s), 0.88 (9 H, s), 0.11 (3 H,s), 0.07 (3 H, s), 0.04 (6 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ217.8,176.6, 164.9, 152.5, 141.7, 132.9, 125.0, 119.0, 115.3, 73.5, 73.3,53.4, 47.0, 40.1, 35.8, 33., 29.8, 27.4, 26.0, 25.9, 24.5, 19.0, 18.1,15.2, 14.3, −4.0, −4.2, −4.2, −4.7.

EXAMPLE 54

Macrolactonization:

DCC (0.150 g, 0.725 mmol), 4-DMAP (0.078 g, 0.64 mmol) and 4-DMAP.HCl(0.110 g, 0.696 mmol) were dissolved in CHCl₃ (80 mL) at 80° C. To thisrefluxing solution was added by syringe pump the hydroxy acid 14D (0.0209, 0.029 mmol) and DMAP (0.010 g) in CHCl₃ (10 mL) over 20 h. Thesyringe needle was placed at the base of the condensor to ensure properaddition. After 20 h, the reaction was cooled to 50° C. and AcOH (0.046mL, 0.812 mmol) was added. After 2 h, the reaction was cooled to rt andwashed with sat NaHCO₃ (30 mL), brine (30 mL) and dried over anhydrousNa₂SO₄. The lactone 15D was purified by flash chromatography on silicagel eluting with hexanes/ethyl acetate (20:1-15:1) to give 0.014 g(75%): IR (film): 2929, 1741, 1696, 1254, 1097 cm⁻¹; ¹H NMR (CDCl₃, 400MHz) δ6.95 (1 H, s), 6.55 (1 H, s), 5.48 (1 H, m), 5.37 (1 H, m), 5.16(1 H, d, J=9.8 Hz), 4.17 (1 H, d, J=8.3 Hz), 4.07 (1 H, t, J=7.2 Hz),3.02 (1 H, t, J=7.2 Hz), 2.77 (1 H, m), 2.70 (3 H, s), 2.64 (2 H, m),2.29 (1 H, m), 2.15 (1 H, m), 2.12 (3 H, s), 1.92 (1 H, m), 1.71 (1 H,m), 1.44 (2 H, m), 1.26 (1 H, m), 1.17 (3 H, s), 1.12 (3 H, s), 1.11 (3H, d, J=7.0 Hz), 0.91 (9 H, s), 0.85 (9 H, S), 0.09 (3 H, s), 0.06 (6 H,S), −0.04 (3 H, s); ¹³C NMR (CDCl₃, 100 MHz) δ215.2, 171.9, 164.5,152.5, 138.0, 133.5, 123.8, 120.0, 116.7, 79.4, 76.2, 72.5, 53.5, 47.4,39.9, 34.5, 31.9, 31.5, 30.2, 27.7, 26.1, 25.9, 24.1, 23.8, 23.1, 22.6,19.2, 18.5, 18.2, 16.3, 14.9, 14.1, −3.7, −4.2, −4.7, −5.2.

EXAMPLE 55

Desmethyldesoxyepothilone A (16D):

To the lactone 15D (0.038 g, 0.056 mmol) in THF (2.0 mL) was addedHF-pyridine (1.0 mL). After 2 h, the reaction was poured into sat NaHCO₃(30 mL) and extracted with CHCl₃ (5×20 mL). The organics were dried overNa₂SO₄. The crude diol 16D was purified by flash chromatography onsilica gel eluting with hexanes/ethyl acetate (3:1-2:1) to give 0.023 g(89%): IR (film): 3501, 2933, 1734, 1684, 1290, 1248, 1045 cm⁻¹; ¹H NMR(CDCl₃, 400 MHz) δ6.95 (1 H, s), 6.59 (1 H, s), 5.40 (2 H, m), 5.23 (1H, dd, J=1.4, 9.5 Hz), 4.38 (1 H, bd, J=11.1 Hz), 3.78 (1 H, t, J=6.9Hz), 3.59 (1 H, bs), 3.47 (1 H, s), 2.99 (1 H, q, J=7.0 Hz), 2.68 (3 H,s), 2.66 (1 H, m), 2.46 (1 H, dd, J=11.4, 14.4 Hz), 2.26 (1 H, dd,J=2.2, 14.4 Hz), 2.22 (1 H, m), 2.06 (3 H, s), 1.96 (1 H, m), 1.49 (3 H,m), 1.35 (3 H, m), 1.30 (3 H, s), 1.15 (3 H, d, J=6.9 Hz), 1.06 (3 H,s); ¹³C NMR (CDCl₃, 100 MHz) δ221.5, 170.3, 165.1,.151.8, 139.1, 132.8,125.2, 119.1, 115.5, 78.4, 72.5, 70.8, 53.8, 42.7, 39.6, 32.3, 31.8,28.3, 26.8, 24.8, 23.1, 19.0, 17.2, 16.0, 11.1.

EXAMPLE 56

Epoxide formation:

Diol 16D (0.008 g, 0.017 mmol) was dissolved in CH₂Cl₂ (1.0 mL) andcooled to −60° C. Dimethyldioxirane (0.06 M, 0.570 mL, 0.0034 mmol) wasthen slowly added. The reaction temperature was slowly warmed to −25° C.After 2 h at −25° C., the volatiles were removed from the reaction at−25° C. under vacuum. The resulting residue was purified by flashchromatography on silica gel eluting with MeOH in CH₂Cl₂ (1%-2%) to givea 1.6:1 mixture of cis epoxides 3D and the diastereomeric cis-epoxide(0.0058 g, 74%). The diastereomeric epoxides were separated bypreparative-TLC eluting with hexanes/ethyl acetate (1:1) after 3elutions to give pure diastereomers:

Beta epoxide3D: IR (film): 3458, 2928, 1737, 1685, 1456, 1261, 1150,1043, 1014cm⁻¹; ¹H NMR (CD₂Cl₂, 500 MHz) δ7.01 (1 H, s), 6.56 (1 H, s),5.35 (1 H, dd, J=2.3, 9.6 Hz), 4.30 (1 H, dd, J=3.0, 5.7 Hz), 3.85 (1 H,m), 3.81 (1 H, d, J=5.7 Hz), 3.42 (1 H, d, J=2.0 Hz), 3.03 (1 H, q,J=6.8 Hz), 2.97 (1 H, m), 2.88 (1 H, m), 2.67 (3 H, s), 2.46 (1 H, dd,J=9.0, 14.5 Hz), 2.33 (1 H, dd, J=2.6, 14.5 Hz), 2.13 (1 H, dt, J=3.0,15.0 Hz), 2.08 (3 H, s), 1.82 (1 H, m), 1.52 (6 H, m), 1.41 (1 H, m),1.33 (3 H, s), 1.21 (4 H, m), 1.12 (3 H, d, J=7.0 Hz), 1.06 (3 H, s);¹³C NMR (CD₂Cl₂, 125 MHz) δ221.9, 170.6, 165.6, 152.2, 138.3, 120.2,116.6, 77.3, 73.4, 69.9, 57.7, 55.3, 43.7, 39.7, 32.6, 32.0, 29.8, 27.2,25.7, 24.7, 22.5, 19.2, 19.0, 15.6, 15.6, 11.5;

Alpha epoxide: IR (film): 3439, 2918, 1735; 1684, 1455, 1262, 1048, 1014cm⁻¹; ¹H NMR (CD₂Cl₂, 500 MHz) δ7.02 (1 H, s), 6.56 (1 H, s), 5.62 (1 H,d, J=8.1 Hz), 4.33 (1 H, dd, J=2.7, 11.0 Hz), 3.85 (1 H, t, J=5.9 Hz),3.27 (1 H, d, J=5.3 Hz), 3.11 (1 H, m), 3.07 (1 H, d, J=7.0 Hz), 3.04 (1H, s), 2.87(1 H, m), 2.68 (3 H, s), 2.46 (1 H, dd, J=11.1, 14.1 Hz),2.35 (1 H, dd, J=2.3, 14.1 Hz), 2.11 (3 H, s), 2.06 (1 H, ddd, J=1.9,4.5, 15.1 Hz), 1.87 (1 H, m), 1.52 (6 H, m), 1.38 (2 H, m), 1.29 (3 H,s), 1.08 (3 H, d, J=6.9 Hz), 1.03 (3 H, s); ¹³C NMR (CD₂Cl₂, 125 MHz)δ222.1, 170.2, 165.3, 152.5, 137.6, 119.7, 116.7, 76.7, 72.9, 70.6,57.1, 55.1, 44.7, 40.0, 32.1, 31.4, 30.0, 26.6, 25.5, 24.7, 21.3, 19.3,18.7, 15.7, 11.5.

EXAMPLE 57

Experimental Data for C-12 Hydroxy Epothilone Analogs

Propyl hydroxy compound 43:

¹H NMR (CDCl₃, 400 MHz) d 6.96 (1 H, s), 6.59 (1 H, s), 5.16-5.23 (2 H,band), 4.28 (1 H, m), 3.72 (1 H, m), 3.63 (2 H, t, J=6.3 Hz), 3.17 (1 H,dq, J=2.2, 0.5 Hz), 3.02 (1 H, s), 2.70 (3 H, s), 2.65 (2 H, m), 2.46 (1H, dd, J=10.9, 14.6 Hz), 2.29 (2 H, m), 1.98-2.09 (6 H, band), 1.60-1.91(6 H, band), 1.35 (3 H, s), 1.33 (3 H, s), 1.18 (3 H, d, J=6.8 Hz), 1.07(3 H, s), 1.01 (3 H, d, J=7.1 Hz); ¹³C NMR (CDCl₃, 100 MHz) d 220.69,170.29, 165.00, 151.81, 141.63, 138.93, 120.64, 118.81, 115.52, 78.53,77.23, 73.93, 71.85, 62.26, 53.63, 41.57, 39.54, 37.98, 32.33, 32.14,31.54, 30.75, 29.67, 23.27, 22.89, 18.92, 17.67, 15.98, 15.74, 13.28; MSe/m 536.2, calc 535.29.

Hydroxy methyl compound 46:

¹H NMR (CDCl₃, 400 MHz) d 6.97 (1 H, s), 6.63 (1 H, s), 5.43 (1 H, dd,J=5.7, 9.1 Hz), 5.24 (1 H, d, J=7.4 Hz), 4.31 (1 H, d, J=9.7 Hz), 4.05(2 H, dd, J=7.3, 31.0 Hz), 3.87 (1 H, bs), 3.69 (1 H, bs), 3.17 (1 H,dd, J=2.0, 6.9 Hz), 3.03 (1 H, s), 2.69 (3 H, s), 2.63 (1 H, m), 2.45 (1H, dd, J=11.2, 14.6 Hz), 2.37 (1 H, m), 2.25 (2 H, m), 2.11 (1 H, m),2.05 (3 H, s), 1.78 (1 H, m), 1.70 (1 H, m), 1.35 (3 H, s), 1.34 (2 H,m), 1.29 (1 H, m), 1.18 (3 H, d, J=6.8 Hz), 1.06 (3 H, s), 1.00 (3 H, d,7.0 Hz); ¹³C NMR (CDCl₃, 100 MHz) d 220.70, 170.16, 165.02, 151.63,141.56, 138.41, 121.33, 118.65, 115.33, 77.74, 77.25, 74.11, 71.37,65.73, 53.86, 41.52, 39.52, 37.98, 31.46, 27.70, 25.10, 22.86, 18.74,17.20, 16.17, 15.63, 13.41.

Discussion

Total Synthesis of (−)-Epothilone A

The first known method for preparing epothilone A (1) is provided bythis invention. Carbons 9 through 11 insulate domains of chiralityembracing carbons 3 through 8 on the acyl side of the macrolactone, andcarbons 12 through 15 on the alkyl side. Transmitting stereochemicalinformation from one of the segments to the other is unlikely. Thus, theapproach taken deals with the stereochemistry of each segmentindividually. In the acyl segment, this strategy required knowledge ofboth the relative and absolute configurations of the“polypropionate-like” network. In the alkyl segment, two possibilitiesemerge. In one instance, the C12-C13 epoxide would be included in theconstruct undergoing merger with the acyl related substructure. In thatcase it would be necessary to secure the relative stereochemicalrelationship of carbons 15, 13 and 12. It was necessary to consider thethe possibility that the epoxide would be deleted from the alkyl-sidemoiety undergoing coupling. This approach would only be feasible if theepoxide could be introduced with acceptable stereocontrol after closureof the macrocycle. The synthesis of compound 4, which contains most ofthe requisite stereochemical information required for the acyl fragment,is described above. This intermediate is prepared by a novel oxidativelyinduced solvolytic cleavage of the cyclopropanopyran 3. Also describedabove is a construct containing the alkyl side coupling partnerembodying the absolute and relative stereochemistry at carbons 15, 13and 12, which differs from the alternative approach set forth below.

In considering the union of the alkyl and acyl domains, severalpotential connection sites were available. At some point, an acylationwould be required to establish an ester (or lactone) bond (see boldarrow 2). Furthermore, an aldol construction was required to fashion aC2-C3 connection. Determining the exact timing of this aldol steprequired study. It could be considered in the context of elongating theC3-C9 construct to prepare it for acylation of the C-15 hydroxyl.Unexpectedly, it was discovered that the macrolide could be closed by anunprecedented macroaldolization. (For a previous instance of a ketoaldehyde macroaldolization, see: C. M. Hayward, et al., J. Am. Chem.Soc., 1993, 115, 9345.) This option is implied by bold arrow 3 in FIG.1(A).

The first stage merger of the acyl and alkyl fragments (see boldarrow 1) posed a difficult synthetic hurdle. It is recognized in the art(P. Bertinato, et al., J. Org. Chem., 1996, 61, 8000; vide infra) thatsignificant resistance is encountered in attempting to accomplish bondformation between carbons 9 and 10 or between carbons 10 and 11, whereinthe epoxide would be included in the alkyl coupling partner. Thesecomplications arose from unanticipated difficulties in fashioning acyland alkyl reactants with the appropriate complementarity for mergeracross either of these bonds. An initial merger between carbons 11 and12 was examined. This approach dictated deletion of the oxirane linkagefrom the O-alkyl coupling partner. After testing several permutations,generalized systems 5 and 6 were examined to enter the first stagecoupling reaction. The former series was to be derived from intermediate4. A de novo synthesis of a usable substrate corresponding togeneralized system 5 would be necessary (FIG. 1(B)).

The steps leading from 4 to 11 are shown in Scheme 2. Protection of thefuture C-7 alcohol (see compound 7) was followed by cleavage of thebenzyl ether and oxidation to aldehyde 8. Elongation of the aldehyde tothe terminal allyl containing fragment 10 proceeded through end ether 9(mixture of E and Z geometrical isomers). Finally, the dithiane linkagewas oxidatively cleaved under solvolytic trapping conditions, givingrise to specific coupling component 11. G. Stork; K. Zhao, TetrahedronLett. 1989, 30, 287.

The synthesis of the alkyl fragment started with commercially available(R)-glycidol 12 which was converted, via its THP derivative 13, toalcohol 14. After cleavage of the tetrahydropyran blocking group, theresultant alcohol was smoothly converted to the methyl ketone 15, asshown. The latter underwent an Emmons-type homologation with phosphineoxide 16. D. Meng et al., J. Org. Chem., 1996, 61, 7998. This Emmonscoupling provided a ca. 8:1 mixture of olefin stereoismoers in favor oftrans-17. The resultant alkyne 17 was then converted, via compound 18 toZ-iodoalkene 19 (see FIG. 4(A)). E. J. Corey et al., J. Am. Chem. Soc.,1985, 107, 713.

The critical first stage coupling of the two fragments was achieved by aB-alkyl Suzuki carbon-carbon bond construction. N. Miyaura et al., J.Am. Chem. Soc., 1989, 111, 314; N. Miyaura and A. Suzuki, Chem. Rev.,1995, 95, 2457. Thus, hydroboration of the pre-acyl fragment 11 wasaccomplished by its reaction with 9-BBN. The resultant mixed boranecross-coupled to iodoolefin 19, under the conditions indicated, to give20 in 71% yield. (FIG. 4(B)) Upon cleavage of the acetal, aldehyde 21was in hand.

The availability of 21 permitted exploration of the strategy in whichthe methyl group of the C-1 bound acetoxy function would serve as thenucleophilic component in a macroaldolization. Cf. C. M. Hayward et al.,supra. Deprotonation was thereby accomplished with potassiumhexamethyldisilazide in THF at −78° C. Unexpectedly, these conditionsgive rise to a highly stereoselective macroaldolization, resulting inthe formation of the C-3 (S)-alcohol 22, as shown. The heavypreponderance of 22 was favored when its precursor potassium aldolate isquenched at ca. 0° C. When the aldolate was protonated at lowertemperature, higher amounts of the C-3 (R) compound were detected. Infact, under some treatments, the C-3 (R) epimer predominates. It istherefore possible to generate highly favorable C-3(R):C-3(S) ratios inanalytical scale quenches. In preparative scale experiments, the ratioof 22 to its C-3 epimer is 6:1.

With compound 22 in ready supply, the subgoal of obtainingdesoxyepothilone (23) was feasible. This objective was accomplished byselective removal of the triphenylsilyl (TPS) group in 22, followed,sequentially, by selective silylation of the C-3 alcohol, oxidation ofthe C-5 alcohol, and, finally, fluoride-induced cleavage of the twosilyl ethers.

Examination of a model made possible by the published crystal structureof epothilone (Höfle et al., supra), suggested that the oxirane isdisposed on the convex periphery of the macrolide. Oxidation of 23 wascarried out with dimethyl dioxirane under the conditions shown. Themajor product of this reaction was (−)epothilone A (1), the identity ofwhich was established by nmr, infrared, mass spectral, optical rotationand chromotaraphic comparisons with authentic material. Höfle et al.,supra. In addition to epothilone A (1), small amounts of a diepoxidemixture, as well as traces of the diastereomeric cis C12-C13 monoepoxide(≧20:1) were detected.

The method of synthesis disclosed herein provides workable, practicalamounts of epothilone A. More importantly, it provides routes tocongeners, analogues and derivatives not available from the naturalproduct itself.

Studies Toward a Synthesis of Epothilone A: Use of Hydropyran Templatesfor the Management of Acyclic Stereochemical Relationships

The synthesis of an enantiomerically pure equivalent of the alkoxysegment (carbons 9-15) was carried out in model studies. The keyprinciple involves transference of stereochemical bias from an(5)-lactaldehyde derivative to an emerging dihydropyrone. The latter, onaddition of the thiazole moiety and disassembly, provides the desiredacyclic fragment in enantiomerically pure form.

Various novel structural features of the epothitones make theirsynthesis challenging. The presence of a thiazole moiety, as well as acis epoxide, and a geminal dimethyl grouping are key problems to beovercome. An intriguing feature is the array of three contiguousmethylene groups which serves to insulate the two functional domains ofthe molecules. The need to encompass such an achiral “spacer element”actually complicates prospects for continuous chirality transfer andseems to call for a strategy of merging two stereochemically committedsubstructures. The present invention provides a synthesis of compound 4A(FIG. 14), expecting that, in principle, such a structure could beconverted to the epothilones themselves, and to related screeningcandidates.

The identification of compound 4A as a synthetic intermediate served asan opportunity to illustrate the power of hydropyran matrices inaddressing problems associated with the control of stereochemistry inacyclic intermediates. The synthesis of dihydropyrones was previouslydisclosed through what amounts to overall cyclocondensation of suitablyactive dienes and aldehydic heterodienophiles. Danishefsky, S. J.Aldrichimica Acta, 1986, 19, 59. High margins of steroselectivity can berealized in assembling (cf. 5A+6A→7A) such matrices (FIG. 13). Moreover,the hydropyran platforms service various stereospecific reactions (eeformalism 7A→8A). Furthermore, the products of these reactions areamenable to ring opening schemes, resulting in the expression of acyciicfragments with defined stereochemical relationships (cf. 8A→9A).Danishefsky, S. J. Chemtracts, 1989, 2, 273.

The present invention provides the application of two such routes forthe synthesis of compound 4A. Route 1, which does not per se involvecontrol of the issue of absolute configuration, commences with the knownaldehyde 10A. Shafiee, A., et al., J. Heterocyclic Chem., 1979, 16,1563; Schafiee, A.; Shahocini, S. J. Heterocyclic Chem., 1989, 26, 1627.Homologation, as shown, provided enal 12A. Cyclocondensation of 12A withthe known diene (Danishefsky, S. J.; Kitahara, T. J. Am. Chem. Soc.,1974, 96, 7807), under BF₃ catalysis, led to racemic dihydropyrone 13A.Reduction of 13A under Luche conditions provided compound 14A. Luche,J.-L. J. Am. Chem. Soc., 1978, 100, 2226. At this point it was feasibleto take advantage of a previously introduced lipase methodology forresolution of glycal derivatives through enzymatically mediated kineticresolution. Berkowitz, D. B. and Danishefsky, S. J. Tetrahedron Lett.,1991, 32, 5497; Berkowitz, D. B.; Danishefsky, S. J.; Schulte, G. K. J.Am. Chem. Soc., 1992, 114, 4518. Thus, carbinol 14A was subjected tolipase 30, in the presence of isopropenyl acetate, following theprescriptions of Wong (Hsu, S.-H., et al., Tetrahedron Lett., 1990, 31,6403)to provide acetate 15A in addition to the enantiomerically relatedfree glycal 16A. Compound 15A was further advanced to the PMB protectedsystem 17A. At this juncture, it was possible to use another reactiontype previously demonstrated by the present inventors. Thus, reaction of17A with dimethyldioxirane (Danishefsky, S. J.; Bilodeau, M. T. Angew.Chem. Int. Ed. Engl., 1996, 35, 1381) generated an intermediate(presumably the corresponding glycal epoxide) which, upon treatment withsodium metaperiodate gave rise to aldehyde formate 18A. Allylation of18A resulted in the formation of carbinol 19A in which the formate esterhad nicely survived. (For a review of allylations, see: Yamamoto, Y.;Asao, N. Chem. Rev. 1993, 93, 2207.) However, 19A was accompanied by itsanti stereoisomer (not shown here) [4:1]. Mesylation of the secondaryalcohol, followed by deprotection (see 19A→20A) and cyclization, asindicated, gave compound 4A.

In this synthesis, only about half of the dihydropyrone was securedthrough the process of kinetic resolution. While, in theory, several ofthe synthetic stratagems considered contemplate use of each enantiomerof 15A to reach epothilone itself, another route was sought to allow forfull enantiomeric convergence. The logic of this route is that thechirality of a “dummy” asymmetric center is communicated to the emergingpyran following previously established principles of tunablediastereoselection in the cyclocondensation reaction. (Danishefsky,supra) Cyclo-condensation of lactaldehyde derivative 21A (Heathcock, C.H., et al., J. Org. Chem., 1980, 45, 3846) with the indicated diene,under ostensible chelation control, afforded 22A. The side chain ethercould then be converted to the methyl ketone 25A as shown (see22A→23A→24A→25A). Finally, an Emmons condensations (for example, see:Lythgoe, B., et al., Tetrahedron Lett., 1975, 3863; Toh, H. T.; Okamura,W. H. J. Org. Chem., 1983, 48, 1414; Baggiolini, E. G., et al., J. Org.Chem., 1986, 51, 3098) of 25A with the phoshphine oxide 26A wastransformed to phosphine oxide 26A according to the procedure describedin Toh, supra) as shown in FIG. 15 gave rise to 27A. (The known2-methyl-4-chloromethylthiazole (see Marzoni, G. J. Heterocyclic Chem.,1986, 23, 577.) A straightforward protecting group adjustment thenafforded the previously encountered 17A. This route illustrates theconcept of stereochemical imprinting through a carbon center whicheventually emerges in planar form after conferring enantioselection tosubsequently derived stereocenters. The use of the dihydropyrone basedlogic for securing the stereochemical elements of the epothilones, aswell as the identification of a possible strategy for macrocyclizationwill be described in the following section.

Studies Toward a Synthesis of Epothilone AL Sterocontrolled Assembly ofthe Acyl Region and Models for Macrocyclization

Ring-forming olefin metathesis has been employed to construct16-membered ring congeners related to epothilone A. A stereospecificsynthesis of the C3-C9 sector of the acyl fragment was achieved byexploiting a novel oxidative opening of a cyclopropanated glycal.

Disclosed in the previous section is a synthesis of the “alkoxy” segmentof epothilone (1) (see compound 2B, FIG. 7) encompassing carbons 10 to21. In this section the synthesis of another fragment encoding thestereochemical information of acyl section carbons 3 to 9. It wasenvisioned that the aldehydo center (C)) of the formal target 3B wouldserve as an attachment site to a nucleophilic construct derived fromcompound 2B (requiring placement of a 2 carbon insert, as suggested inFIG. 7), through either inter- or intramolecular means. In such acontext, it would be necessary to deal independently with thestereochemistry of the secondary alcohol center eventually required atC₃. One of the interesting features of system 38 is the presence ofgeminal methyl groups at carbon 4 (epothilone numbering). Again, use ismade of a dihydropyran strategy to assemble a cyclic matrixcorresponding, after appropriate disassembly, to a viable equivalent ofsystem 3B. The expectation was to enlarge upon the dihydropyran paradigmto include the synthesis of germ-dimethyl containing cyclic and acyclicfragments. The particular reaction type for this purpose is generalizedunder the heading of transformation of 4B→5B (see FIG. 7). Commitment asto the nature of the electrophile E is avoided. Accordingly, thequestion whether a reduction would or would not be necessary in goingfrom structure type 5B to reach the intended generalized target 3B isnot addressed.

The opening step consisted of a stereochemically tuneable version of thediene-aldehyde cyclocondensation reaction (FIG. 8; Danishefsky, S. J.,Aldrichimica Acta, 1986, 19, 59), in this instance drawing uponchelation control in the merger of the readily availableenantiomerically homogenous aldehyde 68 with the previously known diene7B. Danishefsky, S. J., et al., J. Am. Chem. Soc. 1979, 101, 7001.Indeed, as precedent would have it, under the influence of titaniumtetrachloride there was produced substantially a single isomer shown ascompound 88. In the usual and stereochemically reliable way(Danishefsky, S. J., Chemtracts Org. Chem. 1989, 2, 273), thedihydropyrone was reduced to the corresponding glycal, 9B. At thispoint, it was feasible to utilize a directed Simmons-Smith reaction forthe conversion of glycal 9B to cyclopropane 108. Winstein, S.;Sonnenberg, J. J. Am. Chem. Soc., 1961, 83, 3235; Dauben, W. G.;Berezin, G. H. J. Am. Chem. Soc., 1963, 85, 468; Furukawa, J., et al.,Tetrahedron, 1968, 24, 53; For selected examples, see Soeckman, R. K.Jr.: Charette, A. B.; Asberon, T.; Johnston, B. H. J. Am. Chem. Soc,1991, 113, 5337; Timmers, C. M.; Leeuwenurgh, M. A.; Verheijen, J. C.;Van der Marel, G. A.; Van Boom, J. H. Tetrahedron: Asymmetry, 1996, 7,49. This compound is indeed an interesting structure in that itcorresponds in one sense to a cyclopropano version of a C-glycoside. Atthe same time, the cyclopropane is part of a cyclopropylcarbinyl alcoholsystem with attendant possibilities for rearrangement. Wenkert, E., etal., J. Amer. Chem. Soc., 1970, 92, 7428. It was intended to cleave theC-glycosidic bond of the cyclopropane in a fashion which would elaboratethe geminal methyl groups, resulting in a solvent-derived glycoside withthe desired aldehyde oxidation state at C-3 (see hypothesizedtransformation 4B→5B, FIG. 7). In early efforts, the non-oxidativeversion of the projected reaction (i.e. E⁺=H⁺) could not be reduced topractice. Instead, products clearly attributable to the ring expandedsystem 11 were identified. For example, exposure of 10B to acidicmethanol gave rise to an epimeric mixture of seven-memberedmixed-acetals, presumably through the addition of methanol tooxocarbenium ion 118.

However, the desired sense of cyclopropane opening, under the influenceof the ring oxygen, was achieved by subjecting compound 10B to oxidativeopening with N-iodosuccinimide. (For interesting Hg(II)-inducedsolvolyses of cyclopropanes that are conceptually similar to theconversion of 10B to 12B, see: Collum, D. B.; Still, W. C.; Mohamadi, F.J. Amer. Chem. Soc., 1986, 108, 2094; Collum, D. B.; Mohamadi, F.;Hallock, J. S.; J. Amer. Chem. Soc., 1983, 105, 6882. Following thisprecedent, a Hg(II)-induced solvolysis of cyclopropane 10B was achieved,although this transformation proved to be less efficient than thereaction shown in FIG. 8.) The intermediate iodomethyl compound,obtained as a methyl glycoside 12B, when exposed to the action oftri-n-butyltinhydride gave rise to pyran 13B containing the geminalmethyl groups. Protection of this alcohol (see 13B→14B), followed bycleavage of the glycosidic bond, revealed the acyclic dithianederivative 15B which can serve as a functional version of thehypothetical aldehyde 3B.

Possible ways of combining fragments relating to 2B and 3B in a fashionto reach epothilone and congeners thereof were examined. In view of thestudies of Schrock (Schrock, R. R., et al., J. Am. Chem. Soc., 1990,112, 3875) and Grubbs (Schwab, P. et al., Angew. Chem. Int. Ed. Engl.,1995, 34, 2039; Grubbs, R. H.; Miller, S. J. Fu, G. C. Acc. Chem. Res.,1995, 28, 446; Schmalz, H.-G., Angew. Chem. Int. Ed. Engl., 1995, 34,1833) and the disclosure of Hoveyda (Houri, A. F., et al., J. Am. Chem.Soc., 1995, 117, 2943), wherein a complex lactam was constructed in akey intramolecular olefin macrocyclization step through a molybdenummediated intramolecuar olefin in metathesis reaction (Schrock, supra;Schwab, supra), the possibility of realizing such an approach wasconsidered. (For other examples of ring-losing metathesis, see: Martin,S. F.; Chen, H.-J.; Courtney, A. K.; Lia, Y.; Pätzel, M.; Ramser, M N.;Wagman, A. S. Tetrahedron, 1996, 52, 7251; Fürstner, A.; Langemann, K.J. Org. Chem., 1996, 61, 3942.)

The matter was first examined with two model ω-unsaturated acids 16B and17B which were used to acylate alcohol 2B to provide esters 18B and 19B,respectively (see FIG. 9). These compounds did indeed undergo olefinmetathesis macrocyclization in the desired manner under the conditionsshown. In the case of substrate 18B, compound set 20B was obtained as amixture of E- and Z-stereoisomers [ca. 1:1]. Diimide reduction of 20Bwas then conducted to provide homogeneous 22B. The olefin methathesisreaction was also extended to compound 19B bearing geminal methyl groupscorresponding to their placement at C4 of epothilone A. Olefinmetathesis occurred, this time curiously producing olefin 21B as asingle entity in 70% yield (stereochemisty tentatively assigned as Z.)Substantially identical results were obtained through the use ofSchrock's molybdenum alkylidene metathesis catalyst.

As described above, olefin metathesis is therefore amenable to thechallenge of constructing the sixteen membered ring containing both therequired epoxy and thiazolyl functions of the target system. It ispointed out that no successful olefin metathesis reaction has yet beenrealized from seco-systems bearing a full compliment of functionalityrequired to reach epothilone. These negative outcomes may merely reflecta failure to identify a suitable functional group constraint patternappropriate for macrocylization.

The Total Synthesis of Epothilone B: Extension of the Suzuki CouplingMethod

The present invention provides the first total synthesis of epothilone A(1). D. Meng, et al., J. Org. Chem, 1996, 61, 7998 P. Bertinato, et al.,J. Org. Chem, 1996, 61, 8000. A. Balog, et al., Angew. Chem. Int. Ed.Engl., 1996, 35, 2801. 0. Meng, et at., J. Amer. Chem. Soc., 1997, 119,10073. (For a subsequent total synthesis of epothilone A, see: Z. Yang,et al., Angew. Chem. Int. Ed. Engl., 1997, 36, 166.) This synthesisproceeds through the Z-desoxy compound (23) which underwent highlystereoselective epoxidation with 2,2-dimethyldioxirane under carefullydefined conditions to yield the desired β-epoxide. The samemyxobacterium of the genus Sorangium which produces 23 also producesepothilone B (2). The latter is a more potent agent than 23, both inantifungal screens and in cytotoxicity/cell nucleus disintegrationassays. G. Höfle, et at., Angew. Chem. Int. Ed. Engl. 1996, 35, 1567; D.M. Bollag, et al., Cancer Res. 1995, 55, 2325.

An initial goal structure was desoxyepothilone B (2C) or a suitablederivative thereof. Access to such a compound would enable the study ofthe regio- and stereoselectivity issues associated with epoxidation ofthe C12-C13 double bond. A key issue was the matter of synthesizingZ-tri-substituted olefinic precursors of 2C with high margins ofstereoselection. A synthetic route to the disubstituted system (A.Balog, et al., Agnew. Chem. Int. Ed. Engl., 1996, 35, 2801) employed apalladium-mediated B-alkyl Suzuki coupling (N. Miyaura, et al., J. Am.Chem. Soc. 1989, 111, 314. (For a review, see: N. Miyaura, A. Suzuki,Chem. Rev. 1995, 95, 2457) of the Z-vinyl iodide 19 (FIG. 4(A)) withborane 7C derived from hydroboration of compound 11 (FIG. 1(A)) with9-BBN (FIG. 4(B)).)

A preliminary approach was to apply the same line of thinking to reach aZ-tri-substituted olefin (FIG. 17) en route to 2C. Two issues had to beaddressed. First, it would be necessary to devise a method to preparevinyl iodide 8C, the tri-substituted analog of 19. If this goal could beaccomplished, a question remained as to the feasibility of conductingthe required B-alkyl Suzuki coupling reaction to reach aZ-tri-substituted olefin. The realization of such a transformation witha “B-alkyl” (as opposed to a “B-alkenyl” system) at the inter-molecularlevel, and where the vinyl iodide is not of the β-iodoenoate (orβ-iodoenone) genre, was not precedented. (For some close analogies whichdiffer in important details from the work shown here, see: N. Miyaura,et al., Bull. Chem. Soc. Jpn. 1982, 55, 2221; M. Ohba, et al.,Tetrahedron Lett., 1995, 36, 6101; C. R. Johnson, M. P. Braun, J. Am.Chem. Soc. 1993, 115, 11014.)

The synthesis of compound 8C is presented in FIG. 16. The route startedwith -olefin 10C which was prepared by catalytic asymmetric allylationof 9C (G. E. Keck, et al., J. Am. Chem. Soc., 1993, 715, 8467) followedby acetylation. Site-selective dihydroxylation of 10C followed bycleavage of the glycol generated the unstable aldehyde 11C.Surprisingly, the latter reacted with phosphorane 12C (J. Chen, et al.,Tetrahedron Lett., 1994, 35, 2827) to afford the Z-iodide 8C albeit inmodest overall yield. Borane 7C was generated from 11 as describedherein. The coupling of compound 7C and iodide 8C (FIG. 16) could beconducted to produce the pure Z-olefin 13C.

With compound 13C in hand, protocols similar to those employed inconnection with the synthesis of 23 could be used. (A. Balog, et al,Angew. Chem. Int. Ed. Engl., 1996, 35, 2801). Thus, cleavage of theacetal linkage led to aldehyde 14C which was now subjected tomacroaldolization (FIG. 17). The highest yields were obtained bycarrying out the reaction under conditions which apparently equilibratethe C3 hydroxyl group. The 3R isomer was converted to the required 3Sepimer via reduction of its derived C3-ketone (see compound 15C). Thekinetically controlled aldol condensation leading to the natural 3Sconfiguration as discribed in the epothilone A series was accomplished.However, the overall yield for reaching the 35 epimer is better usingthis protocol. Cleavage of the C-5 triphenylsilyl ether was followedsequentially by monoprotection (t-butyldimethylsilyl) of the C3hydroxyl, oxidation at C5 (see compound 16C), and, finally, cleavage ofthe silyl protecting groups to expose the C3 and C7 alcohols (seecompound 2C).

It was found that Z-desoxyepothilone B (2C) undergoes very rapid andsubstantially regio- and stereoselective epoxidation under theconditions indicated (although precise comparisons are not available,the epoxidation of 2C appears to be more rapid and regioselective thanis the case with 23) (A. Balog, et al., Angew. Chem. Int. Ed. Engl.,1996, 35, 2801), to afford epothilone B (2) identical with an authenticsample (¹H NMR, mass spec, IR, [α]_(D)). Accordingly, the presentinvention dislcoses the first total synthesis of epothilone B. Importantpreparative features of the present method include the enantioselectivesynthesis of the trisubstituted vinyl iodide 8C, the palladium-mediatedstereospecific coupling of compounds 7C and 8C to produce compound 13C(a virtually unprecedented reaction in this form), and the amenabilityof Z-desoxyepothilone B (2C) to undergo regio- and stereoselectiveepoxidation under appropriate conditions.

Desmethylepothilone A

Total syntheses of epothilones A and B have not been previouslydisclosed. Balog, A., et al., Angew. Chem., Int. Ed. Engl. 1996,35,2801; Nicolaou, K. C., et al., Angew. Chem., Int. Ed. Engl. 1997, 36,166. Nicolaou, K. C., et al., Angew. Chem., Int. Ed. Engl. 1997, 36,525; Schinzer, D., et al., Angew. Chem., Int. Ed. Engl. 1997, 36, 523.Su, D. -S., et al., Angew. Chem. Int. Ed. Engl. 1997, 36, 757. The modeof antitumor action of the epothilones closely mimics that of Taxol™.Höfle, G., et al., H. Angew. Chem., Int. Ed. Engl. 1996, 35, 1567.Although Taxol™ (paclitaxel) is a clinically proven drug, itsformulation continues to be difficult. In addition, taxol induces themultidrug resistance (MDR) phenotype. Hence, any novel agent that hasthe same mechanism of action as taxol and has the prospect of havingsuperior therapeutic activity warrants serious study. Bollag, D. M., etal., Cancer Res. 1995, 55, 2325.

The present invention provides epothilone analogs that are moreeffective and more readily synthesized than epothilone A or B. Thesyntheses of the natural products provide ample material for preliminarybiological evaluation, but not for producing adequate amounts for fulldevelopment. One particular area where a structural change could bringsignificant relief from the complexities of the synthesis would be inthe deletion of the C8 methyl group from the polypropionate domain (seetarget system 3D). The need to deal with this C8 chiral centercomplicates all of the syntheses of epothilone disclosed thus far.Deletion of the C8 methyl group prompts a major change in syntheticstrategy related to an earlier diene-aldehyde cyclocondensation route.Danishefsky, S. J. Chemtracts 1989, 2, 273; Meng, D., et al., J. Org.Chem. 1996, 61, 7998; Bertinato, P., et al., J. Org. Chem. 1996, 61,8000.

As shown in FIG. 20, asymmetric crotylation (87% ee) of 4D (Brown, H.C.; Bhat, K. S. J. Am. Chem. Soc. 1986, 108, 5919), followed byprotection led to TBS ether 5D. The double bond was readily cleaved togive aldehyde 6D. The aldehyde was coupled to the dianion derived fromt-butyl isobutyrylacetate to provide 7D. The ratio of the C₅₅ (7D):C_(5R) compound (not shown) is ca 10:1. That the Weiler-type β-ketoesterdianion chemistry (Weiler, L. J. Am. Chem. Soc. 1970, 92, 6702.; Weiler,L.; Huckin, S. N. J. Am. Chem. Soc. 1974, 96, 1082) can be conducted inthe context of the isobutyryl group suggested several alternateapproaches for still more concise syntheses. Directed reduction of theC₃ ketone of 7D following literature precedents (Evans, D. A., et al.,J. Org. Chem. 1991, 56, 741), followed by selective silylation of the C₃hydroxyl gave a 50% yield of a 10:1 ratio of the required C_(3S) (seecompound 8D) to C_(3R) isomer (not shown). Reduction with sodiumborohydride afforded a ca. 1:1 mixture of C₃ epimers. The carbinol,produced upon debenzylation, was oxidized to an aldehyde which,following methylenation through a simple Wittig reaction, affordedolefin 9D. Treatment of this compound with TBSOTf provided ester 10Dwhich was used directly in the Suzuki coupling with the vinyl iodide12D.

The hydroboration of 10D with 9-BBN produced intermediate 11D which, oncoupling with the vinyl iodide 12D and in situ cleavage of the TBS esterled to 13D (FIG. 21). After de-acetylation, the hydroxy acid 14D was inhand. Macrolactonization of this compound (Boden, E. P.; Keck, G. E. J.Org. Chem. 1985, 50, 2394) produced 15D which, after desilylation,afforded C₈-desmethyldesoxyepothilone (16D). Finally, epoxidation ofthis compound with dimethyldioxirane produced the goal structure 3D. Thestereoselectivity of epoxidation was surprisingly poor (1.5:1) giventhat epoxidation of desoxyepothilone A occurred with >20:1stereoselectivity. Deletion of the C₈ methyl group appears to shift theconformational distribution of 16D to forms in which the epoxidation bydimethyl dioxirane is less β-selective. It is undetermined whether theeffect of the C₈ methyl on the stereoselectivity of epoxidation bydimethydioxirane and the dramatic reduction of biological activity arerelated.

Compounds 3D and 16D were tested for cytotoxicity in cell cultures andassembly of tubulin in the absence of GTP. Microtubule protein (MTP) waspurified from calf brains by two cycles of temperature dependentassembly and disassembly. Weisenberg, R. C. Science 1972, 177, 1104. Incontrol assembly experiments, MTP (1 mg/mL) was diluted in assemblybuffer containing 0.1 M MES (2-(N-morpholino)ethanesulfonic acid), 1 mMEGTA, 0.5 mM MgCl₂, 1 mM GTP and 3M glycerol, pH 6.6. The concentrationof tubulin in MTP was estimated to be about 85%. Assembly was monitoredspectrophotometrically at 350 nm, 35° C. for 40 min by following changesin turbidity as a measure of polymer mass. Gaskin, F.; Cantor, C. R.;Shelanksi, M. L. J. Mol. Biol. 1974, 89, 737. Drugs were tested at aconcentration of 10 μM, in the absence of GTP. Microtubule formation wasverified by electron microscopy. To determine the stability ofmicrotubules assembled in the presence of GTP or drug, turbidity wasfollowed for 40 min after the reaction temperature was shifted to 4° C.

Cytotoxicity studies showed drastically reduced activity in the8-desmethyl series. Compounds 3D and 16D were approximately 200 timesless active than their corresponding epothilone A counterparts (seeTable 1). Recalling earlier SAR findings at both C₃ and C₅, inconjunction with the findings disclosed herein, the polypropionatesector of the epothilones emerges as a particularly sensitive locus ofbiological function. Su, D.-S., et al., Angew. Chem. Int. Ed. Engl.1997, 36, 757; Meng, D., et al., J. Am. Chem. Soc. 1997, 119.

TABLE 1 Relative efficacy of epothilone compounds against drug-sensitiveand resistant human leukemic CCRF-CEM cell lines.^(a) CCRF-CEMCCRF-CEM/VBL CCRF-CEM/VM₁ Compound IC₅₀ (μM)^(b) IC₅₀ (μM)^(b) IC₅₀(μM)^(b) 16D 5.00 5.75 6.29 3D 0.439 2.47 0.764 epothilone A 0.003 0.0200.003 desoxyepothilone A 0.022 0.012 0.013 epothilone B 0.0004 0.0030.002 desoxyepothilone B 0.009 0.017 0.014 paclitaxel 0.002 3.390 0.002^(a)The cytotoxicities of test compounds were determined by the growthof human lymphoblastic leukemic cells CCRF-CEM, or their sublinesresistant to vinblastine and taxol (CCRF-CEM/VBL) or resistant toetoposide (CCRF-CEM/VM-1). XTT-microculture tetrazolium/formazan assayswere used. ^(b)The IC₅₀ values were calculated from 5-6 concentrationsbased on the median-effect plot using computer software.

Biological Results

In the tables which follow, model system I is desoxyepothilone. Modelsystem 2 has the structure.

wherein R′ and R are H.

Model system 3 has the structure

TABLE 2 Relative Efficacy of Epothilone Compound Against Human LeukemicCCRF-CEM Cell Growth and Against CCRF-CEM MDR Sublines Resistant toTaxol or Etoposide IC₅₀ in μM CCRF- CCRF- COMPOUND CCRF-CEM CEM/VLBCEM/VM-1 EPOTHILONE A NATURAL 0.0035 0.0272 0.0034 EPOTHILONE A 0.00290.0203 0.0034 SYNTHETIC MODEL SYSTEM I [3] 271.7 22.38 11.59 TRIOLANALOG [2] 14.23 6.28 43.93 DESOXY EPOTHILONE [1] 0.002 0.012 0.013TAXOL 0.0023 2.63 0.0030 VINBLASTINE 0.00068 0.4652 0.00068 VP-16(ETOPOSIDE) 0.2209 7.388 34.51 Relative Potency of Epothilone CompoundsAgainst Human Leukemic CCRF Sublines CCRF- CEM/VM₁ CCRF-CEM/VBL (Topo IIgene (MDR Cell Line) mutated cell (Taxol Resistant)- line) (TaxolCCRF-CEM (1143 fold) Sensitive) (Parent (Vinblastine (VP-16 Cell Line)Resistant) resistant) IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ IC₅₀ [IC₅₀ (μM) relative to(μM) relative to (μM) relative to (A) (B) Epothilone A (C) EpothiloneCOMPOUND Epothilone A] (B)/(A)] A (C)/(A)] TAXOL 0.0023 [0.72] 2.63[109.6] (1143)^(a) 0.0030 [0.88] (1.30)^(a) MODEL 271.7 [84906] 22.38[932.5] (0082)^(b) 11.59 [3409] SYSTEM I (0.043)^(b) TRIAL 14.23 [4447]6.28 [261.7] (0.44)^(b) 43.93 [12920] ANALOG (3.09)^(a) DESOXY- 0.022[6.9] 0.012 [0.5] (0.55)^(b) 0.013 [3.82] EPOTHILONE (0.59)^(b) AEPOTHILONE 0.0032 [1] 0.024 [1]  (7.5)^(a) 0.0034 [1] A (1.06)^(a)^(a)(B)/(A) or (C)/(A) ratio >1 indicates fold of resistance whencompared with the parent cell line. ^(b)(B)/(A) or (C)/(A) ratio <1indicates fold of collateral sensitvity when compared with the parentcell line.

As shown in Table 2, CCRF-CEM is the parent cell line. CCRF-CEM/VBL (MDRcell line) is 1143-fold resistant to taxol. CCRF-CEM/VM (Topo II mutatedcell line) only 1.3-fold resistant to taxol.

In terms of relative potency, synthetic Epothilone is roughly the sameas natural Epothilone A. For CCRF-CEM cells, the ordering is:

Taxol=Epothilone A>Desoxy Epothilone A>>Triol Analog>>Model System I

For CCRF-CEM/VBL, the relative potency ordering is:

Desoxy Epothilone A≧Epothilone A>>Taxol>Triol Analog>Model System I

For CCRF-CEM/VM, the relative potency ordering is:

Taxol Epothilone A>Desoxy Epothilone A>>Model System I>Triol Analog

It is concluded that CCRF-CEM/VM cells are collaterally sensitive tocertain epothilone compounds.

TABLE 3 Relative Efficacy of Epothilone Compounds Against The DC-3FHamster Lung Cell Growth and Against DC-3F MDR Sublines ResistantActinomylin D IC₅₀ in μM DC-3F/ COMPOUNDS DC-3F DC-3F/ADII ADXEPOTHILONE A NATURAL 0.00368 0.01241 0.0533 EPOTHILONE A SYNTHETIC0.00354 0.0132 0.070 MODEL SYSTEM I [3] 9.52 3.004 0.972 TRIOL ANALOG[2] 10.32 4.60 4.814 DESOXY EPOTHILONE [1] 0.01061 0.0198 0.042 TAXOL0.09469 3.205 31.98 VINBLASTINE 0.00265 0.0789 1.074 VP-16 (Etoposide)0.03386 0.632 12.06 ACTINOMYCIN-D 0.000058 0.0082 0.486 (0.05816 nm)

Concerning Table 3, experiments were carried out using the cell linesDC-3F (parent hamster lung cells), DC-3F/ADII (moderatemultidrug-resistant (MDR) cells) and DC-3F/ADX (very strong MDR cells).

The relative potency of the compounds are as follows:

DC-3F: Actinomycin D > Vinblastine ≧ Epothilone A (0.0036 μM) > Desoxyepothilone > VP-16 > Taxol (0.09 μM) > Model system I and triol analogDC-3F/ Desoxyepothilone ≧ Epothilone A (0.06 μM) > Actinomycin D ADX: >Model system I > Vinblastine > triol analog > viablastine > taxol (32.0μM)

DC-3F/ADX cells (8379-fold resistant to actinomycin D) are >338 fold(ca. 8379 fold) resistant to Taxol, VP-16, Vinblastine and Actinomycin Dbut <20 fold, resistant to epothilone compounds.

In general, these results are similar to those for CCRF-CEM cells.

TABLE 4 Three Drug Combination Analysis (Based on the Mutually ExclusiveAssumption - Classical Isobologram Method) Drug A: EPOTHILONE B (#8)(μM) Drug B: TAXOL (μM) Drug C: VINBLASTINE (μM) Conditions: CCRF-CEM, 3DRUG COMBINATION, RATIO (A:B:C: 1:5:1); EPOTHILONE + TAXOL +VINBLASTINE; EXPOSURE TIME 72 HRS; XTT ASSAY. Combination Index* Valuesat: Parameters Drug ED50 ED75 ED90 ED95 Dm(IC₅₀) (μM) m r A −000611.71561 .98327 B −00109 2.14723 .98845 C −00061 1.76186 .9919 A + B1.51545 1.38631 1.27199 1.20162 −00146 2.41547 .97168 B + C 1.432431.33032 1.23834 1.18091 .00138 2.35755 .95695 A + C .74395 .68314 .62734.59204 .00045 2.0098 .96232 A + B + C 1.37365 1.32001 1.27255 1.24412.00122 2.11202 .93639 VBI → microtubule depolymerization Taxol →microtubule polymerization Epo-B → microtubule polymerization EpothiloneB and Taxol have a similar mechanism of action (polymerization) butEpothilone B synergizes VBL whereas Taxol antagonizes VBL. Taxol + VBL →Antagonism EpoB + Taxol → Antagonism EpoB + VBL → Synergism EpoB +Taxol + VBL → Antagonism *Combination index values <1, −1, and >1indicate synergism, additive effect, and antagonism, respectively.

TABLE 5 Relative cytotoxicity of epothilone compounds in vitro. IC₅₀ inμM Compounds CCRF-CEM CCRF-CEM/VLB CCRF-CEM/VM-1 VINBLASTINE **** 0.0008(0.00063 ± 0.00008) 0.44 (0.332 ± 0.063)  (52.7X)^(§) 0.00049 (0.00041 ±0.00004)  (0.7X) 0.0006 0.221 0.00039 0.0005 0.336 0.00036 VP-16 0.259(0.293 ± 0.019) 6.02 (10.33 ± 2.87)  (35.3X) 35.05 (34.39 ± 4.73)(117.4X) 0.323 9.20 42.24 0.296 15.76 25.89 TAXOL *** 0.0021 4.14 0.0066#17 * 0.090 0.254 #18 1157.6 >>1 #19 0.959 >>1 #20 * 0.030 0.049 #21 — —#22 * 0.098 0.146 #23 — — #24 *** 0.0078 0.053 #25 * 0.021 0.077 #26 *0.055 0.197 #27 **** 0.0010 0.0072 Epothilone A (Syn) *** 0.0021 0.015Epothilone B (Syn) **** 0.00042 0.0017 * Number of asterisks denotesrelative potency. ^(§)Number in parentheses indicates relativeresistance (fold) when compared with parent cell line.

TABLE 6 Relative potency of epothilone compounds in vitro. IC₅₀ in μMCompounds CCRF-CEM CCRF-CEM/VBL Desoxy Epo. A 1 * 0.022 0.012 2 14.236.28 3 271.7 22.38 4 2.119 43.01 5 >20 35.19 Trans. A 6 0.052 0.035 77.36 9.82 Syn-Epo.-B 8 **** 0.00082 0.0029 Natural B 9 **** 0.000440.0026 Desoxy Epo. B 10 *** 0.0095 0.017 Trans. Epo. B 11 * 0.090 0.26212 0.794 >5 13 11.53 5.63 β-desmethyl desoxy-Epo 14 5.42 5.75β-desmethyl Mix-cis Epo 15 0.96 5.95 β-desmethyl β-Epo 15 0.439 2.47β-demethyl α-Epo 16 7.47 16.48 EPOTHILONE A (Natural) *** 0.0024 (0.0027± 0.0003) 0.0211 (0.020 ± 0.001) (7.4X) 0.0031 0.0189 EPOTHILONE B(Natural) **** 0.00017 0.0017 (7.0X) EPOTHILONE B (Synthetic) 0.000550.0031 (0.00213 ± 0.00055) EPOTHILONE B (Synthetic, larger quantitysynthesis) 0.00033 0.0021 (6.1X) (25.9 mg) IC₅₀ in μM CompoundsCCRF-CEM/VM-1 Desoxy Epo. A 1 0.013 2 43.93 3 11.59 4 2.76 5 98.04Trans. A 6 0.111 7 9.65 Syn-Epo.-B 8 0.0044 Natural B 9 0.0018 DesoxyEpo. B 10 0.014 Trans. Epo. B 11 0.094 12 >5 13 14.46 β-desmethyldesoxy-Epo 14 6.29 β-desmethyl Mix-cis Epo 15 2.55 β-desmethyl β-Epo 150.764 β-demethyl α-Epo 16 0.976 EPOTHILONE A (Natural) 0.006 (0.00613 ±0.0001) 0.00625 (2.3X) EPOTHILONE B (Natural) 0.00077 EPOTHILONE B(Synthetic) 0.0018 (0.00126 ± 0.0003) EPOTHILONE B (Synthetic, largerquantity synthesis) 0.0012 (3.6X) (25.9 mg)

TABLE 7 Relative cytotoxicity of epothilone compounds in vitro. IC₅₀ CEMCEM/VBL epothilone A 0.0029 μM 0.0203 μM desoxyepothilone 0.022 0.012  214.2 6.28  3 271.7 22.4  4 2.1 43.8  5 >20 35.2  6 0.052 0.035  7 7.49.8 synthetic epothilone B 0.00082 0.00293 natural epothilone B 0.000440.00263 desoxyepothilone B 0.0095 0.0169 11 0.090 0.262 12 0.794 >5 1311.53 5.63 14 5.42 5.75 15 0.439 2.47 16 7.47 16.48 17 0.090 0.254 181157.6 >>1 19 0.959 >>1 20 0.030 0.049 21 Not Available — 22 0.098 0.14623 Not Available — 24 0.0078 0.053 25 0.0212 0.077 26 0.0545 0.197 270.0010 0.0072

TABLE 8 Chemotherapeutic Effect of Epothilone B, Taxol & Vinblastine inCB-17 Scid Mice Bearing Human CCRF-CEM and CCRF-CEM/VBL Xenograft¹Average weight change Average tumor volume Tumor Drug² Dose Day 0 Day 7Day 12 Day 17 Day 22 Day 7 Day 12 Day 17 Day 22 CCRF-CEM 0 24.4 +0.2+0.4 +0.1 +0.5 1.0³ 1.00 1.00 1.00 Epo B 0.7⁴ 24.7 −0.1 −0.7 −1.4 +0.31.0 0.53 0.48 0.46 1.0⁵ 25.0 +0.1 −1.5 −2.4 +0.1 1.0 0.46 0.35 0.43Taxol 2.0 25.1 −0.1 −1.1 −1.5 −0.3 1.0 0.39 0.29 0.28 4.0 25.1 −0.2 −1.7−1.9 −0.3 1.0 0.37 0.23 0.19 VBL 0.2 25.9 +0.2 −0.8 −1.5 −0.3 1.0 0.450.25 0.29 0.4 25.0 −0.1 −1.4 −1.8 −0.7 1.0 0.31 0.27 0.30 CCRF-CEM/ 026.3 −0.3 +0.1 −0.3 +0.4 1.0 1.00 1.00 1.00 VBL Epo B 0.7 25.8 +0.1 −0.7−1.0 −0.2 1.0 0.32 0.40 0.33 1.0⁶ 26.0 −0.2 −1.3 −2.1 −0.5 1.0 0.41 0.270.31 Taxol 2.0 26.1 0 −0.9 −1.5 −0.1 1.0 0.60 0.58 0.70 4.0 26.0 0 −1.4−1.6 −0.9 1.0 0.79 0.55 0.41 VBL 0.2 25.9 −0.3 −0.8 −1.4 −0.3 1.0 0.860.66 0.67 0.4 25.9 0 −1.2 −1.8 −0.5 1.0 1.02 0.57 0.62 ¹CCRF-CEM andCCRF-CEM/VBL tumor tissue 50 ul/mouse implanted S.C. on day 0,Treatments i.p., QD on day 7, 8, 9, 10, 14 and 15. There were sevenCB-17 scid male mice in each dose group and control. ²Epo B, epothiloneB; VBL, vinblastine. ³The tumor volumes for each group on day 7 wasabout 1 mm³. The average volumes of CCRF-CEM control group on day 12, 17and 22 were 19, 76 and 171 mm³, and of CCRF-CEM/VBL control group were35, 107 and 278 mm³, respectively. ⁴Two mice died of drug toxicity onday 19 & 20. ⁵Three mice died of drug toxicity on day 18, 19 and 21.⁶One mouse died of drug toxicity on day 17.

In summary, epothilones and taxol have similar modes of action bystabilizing polymerization of microtubules. However, epothilones andtaxol have distinct novel chemical structures.

MDR cells are 1500-fold more resistant to taxol (CCRF-CEM/VBL cells),epothilone A showed only 8-fold resistance and epothilone B showed only5-fold resistance. For CCRF-CEM cells, Epo B is 6-fold more potent thanEpo A and 10-fold more potent than Taxol. Desoxyepothilone B and compd#24 are only 3-4-fold less potent than Taxol and compound #27 is >2-foldmore potent than Taxol. Finally, Taxol and vinblastine showed antagonismagainst CCRF-CEM tumor cells, whereas the combination of EpoB+vinblastine showed synergism.

Relative Cytotoxicity of Epothilones against Human Leukemic Cells inVitro is in the order as follows:

CCRF-CEM Leukemic Cells

Epo B (IC₅₀=0.00035 μM; Rel.Value=1)>VBL(0.00063;1/1.8)>#27(0.0010;1/2.9)>Taxol (0.0021; 1/6)>Epo A(0.0027; 1/7.7)>#24(0.0078; 1/22.3)>#10 (0.0095; 1/27.1)>#25 (0.021;1160)>#1 (0.022; 1/62.8)>#20 (0.030; 1/85.7)>#6 (0.052; 1/149)>#260.055; 1/157)>#17 (0.090; 1/257)>VP-16 (0.29; 1/8.29)>#15 (0.44;1/1257)>#19 (0.96; 1/2943)

CCRF-CEM/VBL MDR Leukemic Cells

Epo B (0.0021; 1/61* [1]**)>#27 (0.0072; 1/20.6)>#1 (0.012; 1/34.3)>#10(0.017; 1/48.6)>Epo A (0.020; 1/57.1 [1/9.5])>#6 (0.035)>#20 (0.049)>#24(0.053)>#25 (0.077)>#22 (0.146)>#26 (0.197)>#17 (0.254)>#11 (0.262)>VBL(0.332; 1/948.6 [1/158.1])>Taxol (4.14; 1/11828 [1/1971.4])>VP-16(10.33; 1/29514 [1/4919])

*Potency in parentheses is relative, to Epo B in CCRF-CEM cells.

**Potency in square brackets is relative to Epo B in CCRF-CEM/VBL MDRcells.

As shown in Table 9, treatment of MX-1 xenograft-bearing nude mice withdesoxyepothilone B (35 mg/kg, 0/10 lethality), taxol (5 mg/kg, 2/10lethality; 10 mg/kg, 2/6 lethality) and adriamycin (2 mg/kg, 1/10lethality; 3 mg/kg, 4/6 lethality) every other day, i.p. beginning day 8for 5 doses resulted in a far better therapeutic effect fordesoxyepothilone B at 35 mg/kg than for taxol at 5 mg/kg and adrimycinat 2 mg/kg with tumor volume reduction of 98%, 53% and 28%,respectively. For the desoxyepothilone B-treated group, 3 out of 10 micewere found with tumor non-detectable on day 18. (See FIG. 46) 18 everyother day for 5 more doses resulted in 5 out of 10 mice with tumordisappearing on day 28 (or day 31). See Table 10. By contrast, theextended treatment with taxol at 5 mg/kg for five more doses resulted incontinued tumor growth at a moderate rate, and 2 out of 10 mice died oftoxicity.

Toxicity studies with daily i.p. doses of desoxyepothilone B (25 mg/kg avery effective therapeutic dose as indicated in earlier experiments) for4 days to six mice resulted in no reduction in average body weight(Table 13; FIG. 47) By contrast, epothilone B (0.6 mg/kg, i.p.) for 4days to eight mice resulted in 33% reduction in average body weight; alleight mice died of toxicity between day 5 and day 7.

TABLE 9 Therapeutic Effect of Desoxyepothilone B, Taxol, and Adriamycinin Nude Mice Bearing Human MX-1 Xenograft^(a) Average Body Weight ChangeAverage Tumor Volume Tumor Dose (g) (T/C) Disappear- Drug (mg/kg) Day 810 12 14 16 18 Day 10 12 14 16 18 Died ance Control  0 24.6 −0.1 +1.0+1.0 +1.3 +1.8 1.00 1.00 1.00 1.00 1.00 0/10 0/10 Desoxyepothilone B 3523.0 −0.1 +0.7 −0.3 −1.7 −1.6 0.42 0.28 0.07 0.04 0.02 0/10 3/10 Taxol 5 24.0 −1.3 −0.8 −1.4 −1.9 −1.8 0.58 0.36 0.34 0.42 0.47 2/10 0/10 1024.3 −1.0 −1.0 −2.3 −3.5 −3.8 0.85 0.40 0.21 0.20 0.12 2/6  1/6 Adriamycin  2^(b) 23.9 +0.3 0 −1.4 −1.9 −2.0 0.94 0.88 1.05 0.69 0.721/10 0/10  3^(c) 22.4 +1.3 −0.2 −1.5 −2.1 −2.3 0.72 0.54 0.56 0.51 0.364/6  0/6  ^(a)MX-1 tissue 100 μl/mouse was implanted s.c on day 0. Everyother day i.p. treatments were given on day 8, 10, 12, 14 and 16. Theaverage tumor volume of control group on day 10, 12, 14, 16 and 18 were78, 151, 372, 739 and 1257 mm³, respectively. ^(b)One mouse died oftoxicity on day 22. ^(c)Four mice died of toxicity on day 24.

TABLE 10 Extended Experiment of Desoxyepothilone B, Taxol, Cisplatin andCyclophophamide in Nude Mice Bearing Human MX-1 Xenograft^(a) AverageBody Weight Change Average Tumor Dose (g) Tumor DisappearanceDisappearance # Drug (mg/kg) Day 8 20 22 24 26 28 Day 20 22 24 26 28Duration (Day) Died Desoxyepo B 40 23.0 −1.7 −2.4 −2.4 −1.4 −1.22/10^(b) 2/10 3/10 5/10 5/10 44 (5/10) 0/10 Taxol 5 24.0 −1.6 −0.3 +0.1−0.6 −0.4 0/10 0/10 0/10 0/10 0/10 2/10 10 No extended test 1/6 on day16 Reappear on day 38 2/6 ^(a)Extended experiment was carried out after5 times injection (on day 8, 10, 12, 14 and 16). Every other day i.p.treatments were given continuously: Desoxyepothilone B and Taxol on day18, 20, 22, 24 and 26; control group mice were sacrificed. ^(b)One ofthe mice tumor reappeared on day 20.

TABLE 11 Toxicity of Epothilone B and Desoxyepothilone B in normal nudemice. Dose and Number Schedule of Disap- Dura- Group (mg/kg) mice Diedpearance tion Control 4 0 Epothilone B^(a) 0.6 QD × 4 8 8Desoxyepothilone B 25 QD × 4 6 0 ^(a)Mice died of toxicity on day 5, 6,6, 7, 7, 7, 7, 7

TABLE 12 Therapeutic Effect of Epothilone B, Desoxyepothilone B andTaxol in B6D2F, Mice Bearing B16 Melanoma^(a) Average Weight ChangeAverage Tumor Volume Dose (g) (T/C) #Mice Drug (mg/kg) Day 0 3 5 7 9 11Day 5 7 9 11 Died Control 0 26.5 −0.2 0 −0.2 0 +1.0 1.00 1.00 1.00 1.00 0/15 Epothilone B 0.4 QD × 6^(b) 27.1 −0.2 −0.6 −1.1 −3.4 −3.9 1.081.07 1.27 1.07 1/8 0.8 QD × 5^(c) 27.0 0 −0.8 −3.1 −4.7 −4.7 0.57 0.890.46 0.21 5/8 Desoxyepothilone B 10 QD × 8 27.0 −0.7 −0.9 −1.1 −1.5 −0.30.23 0.22 0.51 0.28 0/6 20 QD 1-4, 7-8 26.9 −1.3 −2.2 −1.3 −1.6 −0.80.59 0.63 0.58 0.33 0/6 Taxol 4 QD × 8 26.7 +0.1 +0.2 +0.3 +0.4 +0.80.62 0.39 0.56 0.51 0/8 6.5 QD × 8 26.7 +0.1 +0.3 +0.3 +0.4 +1.7 0.190.43 0.20 0.54 0/8 ^(a)B16 melanoma cells 1.2 × 10⁶/mouse was implantedS.C. on day 0. Daily treatments start on day 1 after inoculation. Numberof mice in each group: Control, 15; Epothilone B, 8; Desoxythilone B, 5and Taxol, 8. The average tumor volume of control group on day 5, 7, 9and 11 were 16, 138, 436 and 1207 mm³, respectivety. See FIGS. 44(a) and(b). ^(b)One mouse died of toxicity on day 10. ^(c)Five mice died oftoxicity on day 8, 10, 10, 11, 12. One moribund mouse was sacrificed fortoxicological examinations on day 11.

TABLE 13 Therapeutic Effect of Desoxyepothilone B, Epothilone B, Taxoland Vinblastine in Nude Mice Bearing Human MX-1 Xenograft^(a) AverageBody Weight Change Average Tumor Volume Dose (g) (T/C) Drug (mg/kg) Day7 11 13 15 17 Day 11 13 15 17 Note Control 27.9 +0.8 +1.1 +1.9 +0.6 1.001.00 1.00 1.00 0/8 died Desoxyepothilone B 15 27.1 +0.8 +1.1 +1.6 +1.50.65 0.46 0.49 0.41 0/6 died 25^(b) 27.0 +0.4 +0.7 +1.0 +0.7 0.38 0.110.05 0.04 0/6 died (1/6 cured on day 35) Epothilone B  0.3 26.9 +0.5+0.4 −0.3 −1.2 1.00 0.71 0.71 0.84 0/7 died  0.6^(c) 27.4 −0.3 −1.3 −2.1−2.1 1.08 0.73 0.81 0.74 3/7 died Taxol  5 26.9 −0.1 +0.4 +1.1 +1.2 0.540.46 0.40 0.45 0/7 died 10^(d) 27.6 −2.7 −1.1 −0.3 +2.2 0.43 0.37 0.120.11 4/7 died Vinblastine  0.2 25.7 +0.6 +1.4 +2.3 +2.9 0.65 0.54 0.560.88 0/7 died  0.4^(c) 26.4 +0.8 +0.5 +1.9 +2.1 0.80 0.56 0.83 0.88 1/7died ^(a)MX-1 tissue 50 μl/mouse was implanted s.c. on day 0. Everyother day i.p. treatments were given on day 7, 9, 11, 13 and 15. Numberof mice in each group: Control, 8: Desoxyepothilone B, 6; Epothilone B,7; Taxol, 7 and Vinblastine, 7. The average tumor volume of controlgroup on day 11, 13, 15 and 17 were 386, 915, 1390 and 1903 mm³,respectively. See FIG. 45. ^(b)One out of six mice with no detectabletumor on day 35. ^(c)Three mice died of drug toxicity on day 17. Everyother day i.p. treatments were given except day 15. ^(d)Four mice diedof drug toxicity on day 13, 13, 13, 15. ^(e)One mouse died of drugtoxicity on day 15.

TABLE 14 Toxicity of Hematology and Chemistry of Desoxyepothilone B, andTaxol in Nude Mice Bearing Human MX-1 Xenograft^(a) Hematology^(b) WBCChemistry^(b) Dose Total Neutro- Lymph RBC PLT GOT GPT Drug (mg/kg ip)(10³/mm³) phils (%) (%) (10³/mm³) (10⁶/mm³) (U/L) (U/L) Control 12.9 3861 8.1 800 (n = 4) 203 45 (n = 4) Desoxyepo- 25 and 35^(c) 11.8 48 488.4 700 (n = 6) 296 55 (n = 3) thilone B Taxol  5 and 6^(d) 10.9 51 486.1 1083 (n = 5)  438 79 (n = 5) Normal range^(e) 6.91-12.9 8.25-40.862-90 10.2-12.0 190-340 260 138.7 ^(a)Minced MX-1 tumor tissue 50μl/mouse was implanted s.c. on day 0. ^(b)All assays were determined onday 30; averaged values were given. ^(c)Desoxyepothilone B 25 mg/kg wasgiven i.p. on day 7, 9, 11, 13, 15; 35 mg/kg on day 17, 19, 23, 24, 25.^(d)Taxol 5 mg/kg was given i.p. on day 7, 9, 11, 13, 15; 6 mg/kg on day17, 19, 23, 24, 25. ^(e)Normal ranges are for wild type deer mice andC₃Hej mice (obtained from clinical, biochemical and hematologicalReference values in Normal Experimental Animals, Brtjm Mitruka, ed.,Masson Publishing USA, Inc., N.Y., 1977, and from Clinical Chemistry ofLaboratory Animals, Weter F. Loeb, ed., Pergamon Press, 1989)

TABLE 15 Therapeutic Effect of Desoxyepothilone B, Taxol, Adriamycin,and Camptothecin in Nude Mice Bearing MDR Human MCF-7/Adr Tumor. AverageBody Weight Change Average Tumor Volume Dose (g) (T/C) Drug (mg/kg) Day8 11 13 15 17 Day 11 13 15 17 Died Control 0 25.0 +2.0 +2.6 +3.1 +3.71.00 1.00 1.00 1.00 0/8 DesoxyEpoB 35 25.0 +0.3 +0.7 +0.6 +0.8 0.31 0.270.30 0.34 0/8 TaxoI 6 25.3 +1.7 +1.8 +0.8 +0.9 0.57 0.66 0.85 0.90 0/812 24.5 +0.7 −1.3 −2.4 0 0.50 0.51 0.32 0.40 3/6 Adriamycin 1.5 25.6+0.2 −0.4 −0.6 −0.4 0.70 0.68 0.84 0.78 0/8 3 24.6 +0.5 −1.5 −3.2 −1.60.66 0.83 0.57 0.53 3/6 Camptothecin 1.5 24.4 +1.1 +0.9 +1.7 +1.4 1.080.72 0.61 0.72 0/8 3.0 24.5 −0.6 −0.4 −0.8 −0.9 0.95 0.76 0.61 0.43 0/6^(a)MCF-7/Adr cell 3 × 10⁶/mouse was implanted s.c. on day 0. Everyother day i.p. treatments were given on day 8, 10, 12, 14 and 16. Theaverage tumor volume of control group on day 11, 13, 15 and 17 were 392,919, 1499 and 2372 mm³, respectively.

As evident from Table 15, desoxyepothilone B performs significantlybetter than taxol, vinblastine, adriamycin and camptothecin against MDRtumor xenografts (human mammary adeoncarcinoma MCF-7/Adr xenografts).This drug-resistant tumor grows very aggressively and is refractory totaxol and adriamycin at half their lethal doses. Taxol at 6 mg/kg i.p.Q2Dx5 reduced tumor size only 10% adriamycin resulted in only a 22%reduction on day 17. Whereas, desoxyepothilone B at 35 mg/kg reducedtumor size by 66% on day 17 and yet showed no reduction in body weightor apparent toxicity. Even at the LD₅₀ dosage for taxol (12 mg/kg) oradriamycin (3 mg/kg), desoxyepothilone B still performed moreeffectively. By comparison, camptothecin at 1.5 and 3.0 mg/kg reducedtumor size by 28% and 57%, respectively. Overall, in comparison with thefour important anitcancer drugs in current use, i.e., taxol, adriamycin,vinblastine and camptothecin, desoxyepothilone B showed superiorchemotherapeutic effect against MDR xenografts.

TABLE 16 Extended Experiment of Desoxyepothilone B, Taxol in Nude MiceBearing Human MX-1 Xenograft^(a) Average Body Weight Change AverageTumor Dose (g) Tumor Disappearance Disappearance Drug (mg/kg) Day 8 2022 24 26 28 Day 20 22 24 26 28 Duration (Day) Died Desoxyepo B 40 23.0−1.7 −2.4 −2.4 −1.4 −1.2 2.10^(b) 2/10 3/10 5/10 5/10 44_((5/10)) 0/10Taxol 5 24.0 −1.6 −0.3 +0.1 −0.6 −0.4 0/10 0/10 0/10 0/10 0/10 2/10 10No Extended Test 1/6 on day 16, Reappear on day 38 2/6_((0/8))^(a)Extended experiment was going on after 5 times injection (on day 8,10, 12, 14 and 16). Every other day i.p. treatments were givencontinuously: Desoxyepothilone B and Taxol on day 18, 20, 22, 24 and 26;Control group mice were sacrificed. ^(b)In one of the mice, a tumorreappeared on day 20.

TABLE 17 Therapeutic Effects of Desoxyepothilone B, Taxol in Nude MiceBearing MX-1 Xenograft. # Died CONTROL Treatment Schedule of Day 8 10 1214 16 18 20 toxicity Tumor Size 19 ± 2 78 ± 8 151 ± 15 372 ± 55 739 ±123 1257 ± 184 1991 ± 331 0/10 (mm³) Sacrificed (n = 10)DESOXYEPOTHILONE B Dose Schedule 35 mg/kg on day 40 mg/kg on day NoTreatment Day 8 10 12 14 16 18 20 22 24 26 28 30 45 47 50 60 0/10 TumorSize Mouse 1 15 15 40 40 15 32 30 30 30 30 0 0 0 24 S* — Mouse 2 23 2315 15 15 15 30 48 48 0 30 48 900 1200 S — Mouse 3 15 60 90 105 105 12696 150 180 0 48 64 600 600 S — Mouse 4 21 38 38 0 0 10 8 8 8 8 0 0 0 0 00 Mouse 5 12 23 50 12 0 4 0 0 0 0 0 0 0 0 0 0 Mouse 6 15 40 32 8 8 8 812 12 12 12 30 120 120 S — Mouse 7 21 30 15 15 8 8 8 8 8 8 8 8 180 280 S— Mouse 8 20 48 70 15 15 8 8 0 0 0 0 0 0 8 S — Mouse 9 25 50 40 15 8 0 00 0 0 0 0 0 0 4 4 Mouse 10 20 38 38 38 38 25 25 25 0 0 15 15 100 100 S —TAXOL Dose Schedule 5 mg/kg on day 5 mg/kg on day Day 8 10 12 14 16 1820 22 24 26 28 30 45 47 50 60 2/10 Tumor Size 17 ± 45 ± 54 ± 128 ± 311 ±596 ± 1114 ± 1930 ± 2285 ± S (n = 10) 2 7 13 42 115 151 346 569 597Extended studies → Extended observations → Experiment ended *S:Sacrificed due to tumor burden

TABLE 18 Toxicity of Epothilone B and Desoxyepothilone B in normal nudemice Dose and Schedule Group (mg/kg) Number of mice Died Control 4 0Epothilone B^(a) 0.6 QD × 4 8 8 Desoxyepothilone B 25 QD × 4 6 0^(a)Mice died of toxicity on day, 5,6,6,7,7,7,7,7

What is claimed is:
 1. A compound of formula

wherein X is a halogen; Y is S or O; R₁ is hydrogen, methyl, ethyl,n-propyl, n-hexyl,

—CH₂OTBS, or —(CH₂)₃-OTBDPS; and R₂ is hydrogen, linear or branchedalkyl, alkyoxyalkyl, substituted or unsubstituted aryloxylalkyl,trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linearor branched acyl, substituted or unsubstituted aroyl or benzoyl, orprotecting group.
 2. The compound of claim 1, wherein X is I.
 3. Thecompound of claim 1, wherein X is Br.
 4. The compound of claim 1,wherein R₁ is hydrogen.
 5. The compound of claim 1, wherein R₁ ismethyl.
 6. The compound of claim 1, wherein R₂ is hydrogen.
 7. Thecompound of claim 1, wherein R₂ is acetyl.
 8. The compound of claim 1,wherein R₂ is trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, ortriarylsilyl.
 9. The compound of claim 1, wherein R₂ is TMS.
 10. Thecompound of claim 1, wherein R₂ is TBDMS.
 11. The compound of claim 1,wherein R₂ is Troc.
 12. The compound of claim 1, wherein R₂ is TES. 13.The compound of claim 1, wherein X is I, Y is S, R₁ is methyl, and R₂ isTBDMS.
 14. The compound of claim 1, wherein X is I, Y is S, R₁ ismethyl, and R₂ is acetyl.
 15. The compound of claim 1, wherein X is I, Yis S, R₁ is hydrogen, and R₂ is TBDMS.
 16. The compound of claim 1,wherein X is I, Y is S, R₁ is hydrogen, and R₂ is aetyl.
 17. Thecompound of claim 1, wherein Y is S.
 18. The compound of claim 1,wherein Y is O.
 19. The compound of claim 1, wherein X is I, Y is O, R₁is methyl, and R₂ is TBDMS.
 20. The compound of claim 1, wherein X is I,Y is O, R₁ is methyl, and R₂ is acetyl.
 21. The compound of claim 1,wherein X is I, Y is O, R₁ is methyl, and R₂ is TES.
 22. The compound ofclaim 1, wherein X is I, Y is O, R₁ is hydrogen, and R₂ is TBDMS. 23.The compound of claim 1, wherein X is I, Y is O, R₁ is hydrogen, and R₂is acetyl.
 24. The compound of claim 1, wherein X is I, Y is O, R₁ ishydrogen, and R₂ is TES.
 25. A method of preparing a compound having thestructure:

wherein X is a halogen; Y is S or O; R₁ is hydrogen, methyl, ethyl,n-propyl, n-hexyl,

—CH₂OTBS, or —(CH₂)₃-OTBDPS; and R₂ is hydrogen, linear or branchedalkyl, alkyoxyalkyl, substituted or unsubstituted aryloxylalkyl,trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl, triarylsilyl, linearor branched acyl, substituted or unsubstituted aroyl or benzoyl, orprotecting group, which comprises (a) oxidatively cleaving a compoundhaving the structure:

 using periodate to form an aldehyde intermediate; and (b) condensingthe aldehyde intermediate with a halomethylene transfer agent underbasic conditions to form the desired compound.
 26. The method of claim25, wherein X is iodine.
 27. The method of claim 25, wherein Y is S. 28.The method of claim 25, wherein Y is O.
 29. The method of claim 25,wherein the halomethylene transfer agent is Ph₃P═CR₁I or (Ph₃P⁺CHR₁I)I⁻.30. A method of preparing a haloalkene having the structure:

wherein X is a halogen; Y is S or O; and R₂ is hydrogen, linear orbranched alkyl, alkyoxyalkyl, substituted or unsubstitutedaryloxylalkyl, trialkylsilyl, aryldialkylsilyl, diarylalkylsilyl,triarylsilyl, linear or branched acyl, substituted or unsubstitutedaroyl or benzoyl, or protecting group, which comprises (a) coupling acompound having the structure:

 with a methyl ketone having the structure:

wherein R′ is independently a linear or branched alkyl, alkoxyalkyl,substituted or unsubstituted aryl or benzyl, under suitable conditionsto form a compound having the structure:

(b) treating the compound formed in step (a) under basic conditions toform a Z-iodoalkene having the structure:

 and (c) optionally, deprotecting and acylating the Z-iodoalkene formedin step (b) with N-iodosuccinimide in the presence of a reducing agentto form the desired compound.
 31. The method of claim 30, wherein X isiodine.
 32. The method of claim 30, wherein Y is S.
 33. The method ofclaim 30, wherein Y is O.